Methods of Detecting Bladder Cancer

ABSTRACT

Compositions and methods for detecting bladder cancer are provided. In some embodiments, methods of monitoring recurrence of bladder cancer are provided. In some embodiments, the methods comprise detecting a set of markers consisting of CRH, IGF2, KRT20, and ANXA10.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 14/394,352,filed Oct. 14, 2014, which claims the benefit of U.S. provisionalapplication No. 61/636,194, filed Apr. 20, 2012 and of U.S. provisionalapplication No. 61/770,803 filed Feb. 28, 2013, the entire contanct ofeach is which are incorporated herein by reference in their entirety.

2. SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2019-02-11_01148-0006-01US_Sequence_Listing_ST25” created on Feb. 11,2019, which is 42,704 bytes in size. The information in the electronicformat of the sequence listing is incorporated herein by reference inits entirety.

3. FIELD OF THE INVENTION

Compositions and methods for detecting bladder cancer are provided. Inparticular, bladder cancer markers and panels of markers useful in thedetection of bladder cancer are provided.

4. BACKGROUND

386,000 cases of bladder cancer are diagnosed globally each year,including 70,500 cases per year in the United States. The incidence ofbladder cancer is three times higher in men than in women. The highestincidence and prevalence are found in the European Union, North America,North Africa, and the Middle East

Smoking is the greatest risk factor for bladder cancer. Additional riskfactors include chemical exposure, chemotherapy (such as Cytoxan),radiation treatment, and chronic bladder infection.

Bladder tumors include papillary tumors, which are urothelial carcinomasthat grow narrow, finger-like projections; and nonpapillary (sessile)tumors, such as carcinoma-in-situ, which are less common but have a highrisk of becoming invasive.

Symptoms of bladder cancer can include abdominal pain, blood in theurine, bone pain or tenderness, fatigue, painful urination, frequenturination, urinary urgency, incontinence, and weight loss. Diagnosis isgenerally based on imaging, urinalysis, and/or biopsy.

The prognosis for bladder cancer depends on the stage of cancer atdiagnosis. The prognosis for early tumors is favorable, while theprognosis for advanced tumors is poor. Long-term follow up isrecommended to detect cancer recurrence, which occurs in up to 70% ofbladder cancers. For the first two years, cystoscopy and urine cytologyare recommended every 3 to 4 months, and then at longer intervals insubsequent years, often for the patient's lifetime. These methods areinvasive and costly, making bladder cancer one of the most expensivecancers to treat from diagnosis until death.

Existing non-invasive diagnostic tests include ImmunoCyt™ (Scimedx,Denville, N.J.) and UroVysion® (Abbott Molecular, Abbott Park, Ill.).ImmunoCyt™ is a cytology assay that uses a cocktail of three monoclonalantibodies labeled with fluorescent markers to detect certain cellularmarkers of bladder cancer in exfoliated cells isolated from urinesamples. ImmunoCyt™ is used in conjunction with standard urine cytologyto improve cytology's sensitivity at detecting tumor cells. UroVysion®is also a cytology-based assay, which detects aneupoloidy in certainchromosomes via fluorescent in situ hybridization (FISH). Determinationof the results is conducted by enumerating signals through microscopicexamination of the nucleus of cells in urine.

Improved methods for early detection of bladder cancer are needed. Inparticular, an accurate urine-based diagnostic test that does not relyon cytology could reduce the need for costly and invasive cystoscopy andlabor-intensive and potentially subjective cytology assays.

5. SUMMARY

Compositions and methods for detecting bladder cancer are provided. Inparticular, bladder cancer markers and panels of markers useful in thedetection of bladder cancer are provided. In some embodiments, thelevels of CRH, IGF2, KRT20 and ANXA10 mRNA are measured, for example, byquantitative RT-PCR, and the results can be used to determine whether ornot a subject has bladder cancer. In some embodiments, the levels ofCRH, IGF2, KRT20, and ANXA10 mRNA are normalized to an endogenouscontrol. In some embodiments, the endogenous control is ABL mRNA. Insome embodiments, an endogenous control is selected that is expected tobe expressed at similar levels in bladder urothelial cells from subjectswith and without bladder cancer. In some embodiments, the sample is aurine sample. In some embodiments, the present methods are used tomonitor subjects with a history of bladder cancer for tumor recurrence.In some embodiments, the subject has been treated with BacillusCalmette-Guerin (BCG) within the past three months. In some embodiments,the present methods are used to detect bladder cancer in subjects withno history of bladder cancer. In some such embodiments, the subjectshave symptoms of bladder cancer. Nonlimiting exemplary symptoms ofbladder cancer include abdominal pain, blood in the urine, bone pain ortenderness, fatigue, painful urination, frequent urination, urinaryurgency, incontinence, and weight loss.

In some embodiments, methods for detecting the presence of bladdercancer in a subject are provided. In some embodiments, a methodcomprises detecting the levels of each marker of a set of bladder cancermarkers in a sample from the subject, wherein the set of bladder cancermarkers consists of corticotrophin releasing hormone (CRH), insulin-likegrowth factor 2 (IGF2), keratin 20 (KRT20) and annexin 10 (ANXA10). Insome embodiments, detection of an elevated level of at least one markerindicates the presence of bladder cancer in the subject. In someembodiments, a method further comprises detecting an endogenous control.In some embodiments, the endogenous control is selected from ABL, GUSB,GAPDH, TUBB, and UPK1. In some embodiments, the endogenous control isABL. In some embodiments, a method comprises detecting an exogenouscontrol. In some embodiments, the exogenous control is an RNA. In somesuch embodiments, the exogenous control is an Armored RNA®.

In some embodiments, detecting comprises RT-PCR. In some embodiments,detecting comprises quantitative RT-PCR. In some embodiments, a methodcomprises comparing a Ct value or a ΔCt value to a threshold Ct value orΔCt value. In some embodiments, ΔCt is the Ct value for the endogenouscontrol minus the Ct value for the marker. In some embodiments, theRT-PCR reaction takes less than three hours or less than 2 hours from aninitial denaturation step through a final extension step.

In some embodiments, a method comprises contacting RNA from the samplewith a set of bladder cancer marker primer pairs, wherein the set ofbladder cancer marker primer pairs consists of a first primer pair fordetecting CRH, a second primer pair for detecting IGF2, a third primerpair for detecting KRT20, and a fourth primer pair for detecting ANXA10.In some embodiments, the first primer pair comprises a first primercomprising SEQ ID NO: 19 and a second primer comprising SEQ ID NO: 20,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, thefirst primer pair comprises a first primer comprising SEQ ID NO: 35 anda second primer comprising SEQ ID NO: 36, wherein each primer is lessthan 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the second primer pair comprisesa first primer comprising SEQ ID NO: 16 and a second primer comprisingSEQ ID NO: 17, wherein each primer is less than 50, less than 45, lessthan 40, less than 35, or less than 30 nucleotides long. In someembodiments, the second primer pair comprises a first primer comprisingSEQ ID NO: 32 and a second primer comprising SEQ ID NO: 33, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long. In some embodiments, the third primerpair comprises a first primer comprising SEQ ID NO: 13 and a secondprimer comprising SEQ ID NO: 14, wherein each primer is less than 50,less than 45, less than 40, less than 35, or less than 30 nucleotideslong. In some embodiments, the third primer pair comprises a firstprimer comprising SEQ ID NO: 29 and a second primer comprising SEQ IDNO: 30, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising SEQ ID NO: 26 anda second primer comprising SEQ ID NO: 27, wherein each primer is lessthan 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the fourth primer pair comprisesa first primer comprising SEQ ID NO: 38 and a second primer comprisingSEQ ID NO: 39, wherein each primer is less than 50, less than 45, lessthan 40, less than 35, or less than 30 nucleotides long. In someembodiments, the fourth primer pair comprises a first primer comprisingSEQ ID NO: 48 and a second primer comprising SEQ ID NO: 39, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long.

In some embodiments, a method comprises contacting RNA from the samplewith a set of bladder cancer marker primer pairs, wherein the set ofbladder cancer marker primer pairs consists of a first primer pair fordetecting CRH, a second primer pair for detecting IGF2, a third primerpair for detecting KRT20, and a fourth primer pair for detecting ANXA10.In some embodiments, the first primer pair comprises a first primercomprising at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, or at least 15, at least 16, at least 17,or at least 18 nucleotides of SEQ ID NO: 19 and a second primercomprising at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, or at least 15, at least 16, at least 17,or at least 18 nucleotides of SEQ ID NO: 20, wherein each primer is lessthan 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the first primer pair comprises afirst primer comprising at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, or at least 15, at least 16,at least 17, or at least 18 nucleotides of SEQ ID NO: 35 and a secondprimer comprising at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 36, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long. In some embodiments, the second primerpair comprises a first primer comprising at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 16 and a second primer comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15,at least 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 17,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, thesecond primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 32 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 33, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thethird primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 13 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 14, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thethird primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 29 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 30, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 26 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 27, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 38 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 39, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 48 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 39, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long.

In some embodiments, a method further comprises contacting RNA from thesample with an endogenous control primer pair. In some such embodiments,the endogenous control primer pair is for detecting an endogenouscontrol is selected from ABL, GUSB, GAPDH, TUBB, and UPK1. In someembodiments, the endogenous control primer pair is for detecting ABL. Insome embodiments, the endogenous control primer pair comprises a firstprimer comprising SEQ ID NO: 8 and a second primer comprising SEQ ID NO:9, wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, theendogenous control primer pair comprises a first primer comprising SEQID NO: 41 and a second primer comprising SEQ ID NO: 42, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long. In some embodiments, the endogenouscontrol primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 8 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 9, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, theendogenous control primer pair comprises a first primer comprising atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, or at least 15, at least 16, at least 17, or at least 18nucleotides of SEQ ID NO: 41 and a second primer comprising at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 42, wherein each primer is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long.

In some embodiments, a method comprises contacting RNA from the samplewith an exogenous control primer pair. In some such embodiments, theexogenous control primer pair is for detecting an exogenous RNA. In someembodiments, the exogenous control primer pair comprises a first primercomprising SEQ ID NO: 23 and a second primer comprising SEQ ID NO: 24,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, theexogenous control primer pair comprises a first primer comprising SEQ IDNO: 44 and a second primer comprising SEQ ID NO: 45, wherein each primeris less than 50, less than 45, less than 40, less than 35, or less than30 nucleotides long. In some embodiments, the exogenous control primerpair comprises a first primer comprising at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 23 and a second primer comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15,at least 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 24,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, theexogenous control primer pair comprises a first primer comprising atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, or at least 15, at least 16, at least 17, or at least 18nucleotides of SEQ ID NO: 44 and a second primer comprising at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 45, wherein each primer is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long.

In some embodiments, the method comprises forming a set of bladdercancer marker amplicons, wherein the set of bladder cancer markeramplicons consists of a CRH amplicon, an IGF2 amplicon, a KRT20amplicon, and an ANXA10 amplicon, and contacting the bladder cancermarker amplicons with a set of bladder cancer marker probes, wherein theset of bladder cancer marker probes consists of a first probe fordetecting the CRH amplicon, a second probe for detecting the IGF2amplicon, a third probe for detecting the KRT20 amplicon, and a fourthprobe for detecting the ANXA10 amplicon. In some embodiments, the firstprobe comprises SEQ ID NO: 21 or SEQ ID NO: 37, wherein the first probeis less than 50, less than 45, less than 40, less than 35, or less than30 nucleotides long. In some embodiments, the second probe comprises SEQID NO: 34 or SEQ ID NO: 18, wherein the second probe is less than 50,less than 45, less than 40, less than 35, or less than 30 nucleotideslong. In some embodiments, the third probe comprises SEQ ID NO: 15 orSEQ ID NO: 31, wherein the third probe is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long. In someembodiments, the fourth probe comprises SEQ ID NO: 28 or SEQ ID NO: 40,wherein the fourth probe is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefirst probe comprises at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 21 or at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 37, wherein the first probe is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long. In someembodiments, the second probe comprises at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15,at least 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 34 orat least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, or at least 15, at least 16, at least 17, or at least18 nucleotides of SEQ ID NO: 18, wherein the second probe is less than50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the third probe comprises atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, or at least 15, at least 16, at least 17, or at least 18nucleotides of SEQ ID NO: 15 or at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, or at least 15, atleast 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 31,wherein the third probe is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth probe comprises at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 28 or at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 40, wherein the fourth probe is less than 50, less than45, less than 40, less than 35, or less than 30 nucleotides long. Insome embodiments, each bladder cancer marker probe comprises a dye, andwherein each dye is detectably different from the other three labels. Insome embodiments, each bladder cancer marker probe comprises afluorescent dye and a quencher molecule.

In some embodiments, a method comprises forming an endogenous controlamplicon, and contacting the endogenous control amplicon with anendogenous control probe. In some embodiments, the endogenous controlprobe comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 10, 11, 12, or 43,wherein the endogenous control probe is less than 50, less than 45, lessthan 40, less than 35, or less than 30 nucleotides long. In someembodiments, the endogenous control probe comprises a dye that isdetectably different from the dyes of the bladder cancer marker probes.

In some embodiments, a method comprises forming an exogenous controlamplicon, and contacting the exogenous control amplicon with anexogenous control probe. In some embodiments, the exogenous controlprobe comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 25 or 46, wherein theexogenous control probe is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, theexogenous control probe comprises a dye that is detectably differentfrom the dyes of the bladder cancer marker probes and the endogenouscontrol probe.

In some embodiments, the set of bladder cancer markers are detected in asingle multiplex reaction.

In some embodiments, the sample comprises urothelial cells. In someembodiments, the sample is selected from a urine sample and a bladderwashing sample. In some embodiments, the subject has a history ofbladder cancer. In some embodiments, the subject is being monitored forrecurrence of bladder cancer.

In some embodiments, compositions are provided. In some embodiments, acomposition comprises a set of bladder cancer marker primer pairs,wherein the set of bladder cancer marker primer pairs consists of afirst primer pair for detecting CRH, a second primer pair for detectingIGF2, a third primer pair for detecting KRT20, and a fourth primer pairfor detecting ANXA10. In some embodiments, the first primer paircomprises a first primer comprising SEQ ID NO: 19 and a second primercomprising SEQ ID NO: 20, wherein each primer is less than 50, less than45, less than 40, less than 35, or less than 30 nucleotides long. Insome embodiments, the first primer pair comprises a first primercomprising SEQ ID NO: 35 and a second primer comprising SEQ ID NO: 36,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, thesecond primer pair comprises a first primer comprising SEQ ID NO: 16 anda second primer comprising SEQ ID NO: 17, wherein each primer is lessthan 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the second primer pair comprisesa first primer comprising SEQ ID NO: 32 and a second primer comprisingSEQ ID NO: 33, wherein each primer is less than 50, less than 45, lessthan 40, less than 35, or less than 30 nucleotides long. In someembodiments, the third primer pair comprises a first primer comprisingSEQ ID NO: 13 and a second primer comprising SEQ ID NO: 14, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long. In some embodiments, the third primerpair comprises a first primer comprising SEQ ID NO: 29 and a secondprimer comprising SEQ ID NO: 30, wherein each primer is less than 50,less than 45, less than 40, less than 35, or less than 30 nucleotideslong. In some embodiments, the fourth primer pair comprises a firstprimer comprising SEQ ID NO: 26 and a second primer comprising SEQ IDNO: 27, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising SEQ ID NO: 38 anda second primer comprising SEQ ID NO: 39, wherein each primer is lessthan 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the fourth primer pair comprisesa first primer comprising SEQ ID NO: 48 and a second primer comprisingSEQ ID NO: 39, wherein each primer is less than 50, less than 45, lessthan 40, less than 35, or less than 30 nucleotides long.

In some embodiments, a composition comprises a set of bladder cancermarker primer pairs, wherein the set of bladder cancer marker primerpairs consists of a first primer pair for detecting CRH, a second primerpair for detecting IGF2, a third primer pair for detecting KRT20, and afourth primer pair for detecting ANXA10. In some embodiments, the firstprimer pair comprises a first primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 19 and a second primer comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15,at least 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 20,wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, thefirst primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 35 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 36, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thesecond primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 16 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 17, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thesecond primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 32 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 33, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thethird primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 13 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 14, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thethird primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 29 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 30, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 26 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 27, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 38 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 39, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thefourth primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 48 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 39, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long.

In some embodiments, a composition further comprises a set of bladdercancer marker probes, wherein the set of bladder cancer marker probesconsists of a first probe for detecting a CRH amplicon, a second probefor detecting an IGF2 amplicon, a third probe for detecting a KRT20amplicon, and a fourth probe for detecting an ANXA10 amplicon. In someembodiments, the first probe comprises SEQ ID NO: 21 or SEQ ID NO: 37,wherein the first probe is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thesecond probe comprises SEQ ID NO: 34 or SEQ ID NO: 18, wherein thesecond probe is less than 50, less than 45, less than 40, less than 35,or less than 30 nucleotides long. In some embodiments, the third probecomprises SEQ ID NO: 15 or SEQ ID NO: 31, wherein the third probe isless than 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the fourth probe comprises SEQ IDNO: 28 or SEQ ID NO: 40, wherein the fourth probe is less than 50, lessthan 45, less than 40, less than 35, or less than 30 nucleotides long.In some embodiments, the first probe comprises at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 21 or at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, or at least 15, at least 16, at least 17, orat least 18 nucleotides of SEQ ID NO: 37, wherein the first probe isless than 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the second probe comprises atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, or at least 15, at least 16, at least 17, or at least 18nucleotides of SEQ ID NO: 34 or at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, or at least 15, atleast 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 18,wherein the second probe is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, thethird probe comprises at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, or at least 15, at least 16, atleast 17, or at least 18 nucleotides of SEQ ID NO: 15 or at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 31, wherein the third probe is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long. In someembodiments, the fourth probe comprises at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, or at least 15,at least 16, at least 17, or at least 18 nucleotides of SEQ ID NO: 28 orat least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, or at least 15, at least 16, at least 17, or at least18 nucleotides of SEQ ID NO: 40, wherein the fourth probe is less than50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, each bladder cancer marker probecomprises a dye, and wherein each dye is detectably different from theother three labels. In some embodiments, each bladder cancer markerprobe comprises a fluorescent dye and a quencher molecule.

In some embodiments, a composition further comprises an endogenouscontrol primer pair for detecting an endogenous control. In someembodiments, the endogenous control is selected from ABL, GUSB, GAPDH,TUBB, and UPK1. In some embodiments, the endogenous control is ABL. Insome embodiments, the endogenous control primer pair comprises a firstprimer comprising SEQ ID NO: 8 and a second primer comprising SEQ ID NO:9, wherein each primer is less than 50, less than 45, less than 40, lessthan 35, or less than 30 nucleotides long. In some embodiments, theendogenous control primer pair comprises a first primer comprising SEQID NO: 41 and a second primer comprising SEQ ID NO: 42, wherein eachprimer is less than 50, less than 45, less than 40, less than 35, orless than 30 nucleotides long. In some embodiments, the endogenouscontrol primer pair comprises a first primer comprising at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 8 and a second primer comprising at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, or atleast 15, at least 16, at least 17, or at least 18 nucleotides of SEQ IDNO: 9, wherein each primer is less than 50, less than 45, less than 40,less than 35, or less than 30 nucleotides long. In some embodiments, theendogenous control primer pair comprises a first primer comprising atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, or at least 15, at least 16, at least 17, or at least 18nucleotides of SEQ ID NO: 41 and a second primer comprising at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 42, wherein each primer is less than 50, less than 45,less than 40, less than 35, or less than 30 nucleotides long.

In some embodiments, a composition further comprises an endogenouscontrol probe for detecting an endogenous control amplicon. In someembodiments, the endogenous control is selected from ABL, GUSB, GAPDH,TUBB, and UPK1. In some embodiments, the endogenous control is ABL. Insome embodiments, the endogenous control probe comprises at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, or at least 15, at least 16, at least 17, or at least 18 nucleotidesof SEQ ID NO: 10, 11, 12, or 43, wherein the endogenous control probe isless than 50, less than 45, less than 40, less than 35, or less than 30nucleotides long. In some embodiments, the endogenous control probecomprises a dye that is detectably different from the dyes of thebladder cancer marker probes.

In some embodiments, a composition is a lyophilized composition. In someembodiments, the composition is a solution. In some embodiments, thecomposition further comprises urothelial cells. In some embodiments, theurothelial cells are from a urine sample.

Further embodiments and details of the inventions are described below.

6. DETAILED DESCRIPTION 6.1. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “detect”, “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “detectably different” refers to a set oflabels (such as dyes) that can be detected and distinguishedsimultaneously.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to a human. In some embodiments, the methodsdescribed herein may be used on samples from non-human animals.

As used herein, “bladder cancer” is a tumor, such as a transitional cellcarcinoma, arising from the lining of the bladder, and includes lowgrade and high grade bladder cancers, as well as metastatic bladdercancer. “Low grade bladder cancer” refers to superficial tumors thatproject into the interior of the bladder cavity. Low grade bladdercancers have a high rate of recurrence. “High grade bladder cancer”refers to a fast-growing and/or invasive tumor that invades the bladderwall. High grade bladder cancers have the potential to spread (i.e.,metastasize) to other areas of the body. “Metastatic bladder cancer”refers to invasive bladder cancer that has spread to one or morelocations in the body beyond the bladder.

As used herein, the terms “oligonucleotide,” “polynucleotide,” “nucleicacid molecule,” and the like, refer to nucleic acid-containingmolecules, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “oligonucleotide,” refers to a single-strandedpolynucleotide having fewer than 500 nucleotides. In some embodiments,an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to75, or 12 to 50 nucleotides long. Oligonucleotides may be referred to bytheir length, for example, a 24 residue oligonucleotide may be referredto as a “24-mer.”

As used herein, the term “complementary” to a target RNA (or targetregion thereof), and the percentage of “complementarity” of the probesequence to that of the target RNA sequence is the percentage “identity”to the sequence of target RNA or to the reverse complement of thesequence of the target RNA. In determining the degree of“complementarity” between probes used in the compositions describedherein (or regions thereof) and a target RNA, such as those disclosedherein, the degree of “complementarity” is expressed as the percentageidentity between the sequence of the probe (or region thereof) andsequence of the target RNA or the reverse complement of the sequence ofthe target RNA that best aligns therewith. The percentage is calculatedby counting the number of aligned bases that are identical as betweenthe 2 sequences, dividing by the total number of contiguous nucleotidesin the probe, and multiplying by 100. When the term “complementary” isused, the subject oligonucleotide is at least 90% complementary to thetarget molecule, unless indicated otherwise. In some embodiments, thesubject oligonucleotide is at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the target molecule.

A “primer” or “probe” as used herein, refers to an oligonucleotide thatcomprises a region that is complementary to a sequence of at least 8contiguous nucleotides of a target nucleic acid molecule, such as anmRNA or a DNA reverse-transcribed from an mRNA. In some embodiments, aprimer or probe comprises a region that is complementary to a sequenceof at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, or atleast 30 contiguous nucleotides of a target molecule. When a primer orprobe comprises a region that is “complementary to at least x contiguousnucleotides of a target molecule,” the primer or probe is at least 95%complementary to at least x contiguous nucleotides of the targetmolecule. In some embodiments, the primer or probe is at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to thetarget molecule.

A “sample,” as used herein, includes urine samples (including samplesderived from urine samples), and other types of human samples. As usedherein, urine samples include, but are not limited to, whole urine, asample comprising cells from a urine sample, a sample comprising thecell pellet isolated by centrifugation of a urine sample, a samplecomprising cells isolated by filtration of a urine sample, and the like.In some embodiments, a urine sample comprises a preservative, such as apreservative that causes damage, such as lysis, of red and/or whiteblood cells. In some embodiments, a sample is a human sample other thana urine sample, such as a tissue sample (including a bladder tissueand/or bladder tumor sample), a blood sample (including whole blood,serum, plasma, etc.), etc. In some embodiments, a sample is a bladderwashing sample.

As used herein, “corticotrophin releasing hormone” or “CRH” refers to anmRNA that encodes CRH, as well as the CRH protein. In some embodiments,CRH is human CRH. Nonlimiting exemplary human CRH mRNA sequences arefound at GenBank Accession No. NM_000756, and at SEQ ID NO: 1.

As used herein, “insulin-like growth factor 2” or “IGF2” refers to anmRNA that encodes IGF2, as well as the IGF2 protein. In someembodiments, IGF2 is human IGF2. Nonlimiting exemplary human IGF2 mRNAsequences are shown in SEQ ID NOs: 2 to 4.

As used herein, “keratin 20” or “KRT20” refers to an mRNA that encodesKRT20, as well as the KRT20 protein. In some embodiments, KRT20 is humanKRT20. Nonlimiting exemplary human KRT20 mRNA sequences are found atGenBank Accession No. NM_019010, and at SEQ ID NO: 5.

As used herein, “annexin A10” or “ANXA10” refers to an mRNA that encodesANXA10, as well as the ANXA10 protein. In some embodiments, ANXA10 ishuman ANXA10. Nonlimiting exemplary human ANXA10 mRNA sequences arefound at GenBank Accession No. NM_007193, and at SEQ ID NO: 6.

An “endogenous control,” as used herein refers to a moiety that isnaturally present in the sample to be used for detection, and which canbe used to normalize the levels of the bladder cancer markers describedherein (including, but not limited to, CRH, IGF2, KRT20 and ANXA10).Thus, an endogenous control is typically a moiety that is present atsimilar levels from cell to cell, and at similar levels in cells fromsubjects with bladder cancer and cells from subjects without bladdercancer. In some embodiments, an endogenous control is an RNA (such as anmRNA, tRNA, ribosomal RNA, etc.). Nonlimiting exemplary endogenouscontrols include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPK1amRNA. Nonlimiting exemplary human ABL mRNA sequences are found atGenBank Accession No. NM_007313, and at SEQ ID NO: 7. In someembodiments, an endogenous control is selected that can be detected inthe same manner as the bladder cancer markers are detected and, in someembodiments, simultaneously with the bladder cancer markers.

An “exogenous control,” as used herein, refers to a moiety that is addedto a sample to be used for detection. An exogenous control is typicallyselected that is not expected to be present in the sample to be used fordetection, or is present at very low levels in the sample such that theamount of the moiety naturally present in the sample is eitherundetectable or is detectable at a much lower level than the amountadded to the sample as an exogenous control. In some embodiments, anexogenous control comprises a nucleotide sequence that is not expectedto be present in the sample type used for detection of the bladdercancer markers. In some embodiments, an exogenous control comprises anucleotide sequence that is not known to be present in the species fromwhom the sample is taken. In some embodiments, an exogenous controlcomprises a nucleotide sequence from a different species than thesubject from whom the sample was taken. In some embodiments, anexogenous control comprises a nucleotide sequence that is not known tobe present in any species. In some embodiments, an exogenous control isselected that can be detected in the same manner as the bladder cancermarkers are detected and, in some embodiments, simultaneously with thebladder cancer markers. In some embodiments, an exogenous control is anRNA. In some embodiments, an RNA is an Armored RNA®, which comprises RNApackaged in a bacteriophage protective coat. See, e.g., WalkerPeach etal., Clin. Chem. 45:12: 2079-2085 (1999).

In the sequences herein, “U” and “T” are used interchangeably, such thatboth letters indicate a uracil or thymine at that position. One skilledin the art will understand from the context and/or intended use whethera uracil or thymine is intended and/or should be used at that positionin the sequence. For example, one skilled in the art would understandthat native RNA molecules typically include uracil, while native DNAmolecules typically include thymine. Thus, where an RNA sequenceincludes “T”, one skilled in the art would understand that that positionin the native RNA is likely a uracil.

In the present disclosure, “a sequence selected from” encompasses both“one sequence selected from” and “one or more sequences selected from.”Thus, when “a sequence selected from” is used, it is to be understoodthat one, or more than one, of the listed sequences may be chosen.

In the present disclosure, a method that comprises detecting a “a set ofbladder cancer markers consisting of . . . ” involves detection of onlythe bladder cancer markers of the set, and not any further bladdercancer markers. The method may comprise additional components or steps,however, such as detecting endogenous and/or exogenous controls.Similarly, a method or composition that comprises “a set of bladdercancer marker primer pairs” and/or “a set of bladder cancer markerprobes” can include primer pairs and/or probes for only the bladdercancer markers of the set, and not for any other bladder cancer markers.The method or composition may comprise additional components, however,such as one or more endogenous control primer pairs and/or one or moreexogenous control primer pairs.

6.2. Detecting Bladder Cancer

The present inventors have developed an assay for detecting bladdercancer that involves detecting just four markers, CRH, IGF2, KRT20 andANXA10 (plus one or two controls). The presently described assays haveseveral advantages over existing and previously described diagnosticsfor bladder cancer. For example, the present assays do not rely oncytology, which can be costly, and requires trained cytologists foraccurate interpretation of results. Instead, the present assays rely onthe polymerase chain reaction (PCR), and can be carried out in asubstantially automated manner, for example, using the GeneXpert® system(Cepheid, Sunnyvale, Calif.). Mengual et al., Clin Cancer Res (2010) 16:2624-2633, recently described a “12+2 gene expression signature,” whichdetects 12 to 14 genes in an array format. Surprisingly, the presentinventors have found that a much smaller signature, of just four markers(plus one or two controls), is sufficient to provide equivalent orbetter sensitivity and/or specificity over existing diagnostic assaysfor bladder cancer, including the Mengual et al. assay. Further, becausethe present assays use just four markers (plus one or two controls),they can be carried out in a single reaction mixture, using 4 to 6detectably different dyes, such as fluorescent dyes. The present assayscan be completed in under 3 hours, and in some embodiments, under 2hours, using an automated system, for example, the GeneXpert® system.Existing tests can require several days for a laboratory to complete andsend results. In addition, the present assay can be carried out on muchsmaller volumes of urine (in some embodiments, 5 ml or less). Thus, thepresent assays, which rely on PCR of just four markers (plus one or twocontrols) rather than cytology, allows for a fast, one-pot reaction fordiagnosis of bladder cancer, which in many instances can be carried outat the point of care using an automated system such as GeneXpert®.

6.2.1. General Methods

Compositions and methods for detecting bladder cancer are provided. Insome embodiments, compositions and methods for detecting low gradebladder cancer are provided. In some embodiments, compositions andmethods of detecting high grade bladder cancer are provided. In someembodiments, compositions and methods for monitoring the recurrence ofbladder cancer are provided.

In some embodiments, a method of detecting bladder cancer comprisesdetecting the levels of CRH, IGF2, KRT20 and ANXA10. In someembodiments, a method of detecting bladder cancer comprises detectingthe levels of a set of markers consisting of CRH, IGF2, KRT20 andANXA10. In some embodiments, a method of detecting bladder cancercomprises detecting the levels of CRH, IGF2, KRT20 and ANXA10, and atleast one endogenous control. In some embodiments, a method of detectingbladder cancer comprises detecting the levels of a set of markersconsisting of CRH, IGF2, KRT20 and ANXA10, and at least one endogenouscontrol. In some embodiments, a method of detecting bladder cancercomprises detecting the levels of CRH, IGF2, KRT20 and ANXA10, and atleast one exogenous control. In some embodiments, a method of detectingbladder cancer comprises detecting the levels of a set of markersconsisting of CRH, IGF2, KRT20 and ANXA10, and at least one exogenouscontrol. In some embodiments, a method of detecting bladder cancercomprises detecting the levels of CRH, IGF2, KRT20 and ANXA10, at leastone endogenous control, and at least one exogenous control. In someembodiments, a method of detecting bladder cancer comprises detectingthe levels of a set of markers consisting of CRH, IGF2, KRT20 andANXA10, at least one endogenous control, and at least one exogenouscontrol.

In some embodiments, a method of detecting bladder cancer comprisesdetecting the levels of CRH, IGF2, KRT20 and ANXA10 mRNA. In someembodiments, a method of detecting bladder cancer comprises detectingthe levels of a set of markers consisting of CRH, IGF2, KRT20 and ANXA10mRNA. In some embodiments, a method of detecting bladder cancercomprises detecting the levels of CRH, IGF2, KRT20 and ANXA10 mRNA, andat least one endogenous control RNA. In some embodiments, a method ofdetecting bladder cancer comprises detecting the levels of a set ofmarkers consisting of CRH, IGF2, KRT20 and ANXA10 mRNA, and at least oneendogenous control RNA. In some embodiments, a method of detectingbladder cancer comprises detecting the levels of CRH, IGF2, KRT20 andANXA10 mRNA, and at least one exogenous control RNA. In someembodiments, a method of detecting bladder cancer comprises detectingthe levels of a set of markers consisting of CRH, IGF2, KRT20 and ANXA10mRNA, and at least one exogenous control RNA. In some embodiments, amethod of detecting bladder cancer comprises detecting the levels ofCRH, IGF2, KRT20 and ANXA10 mRNA, at least one endogenous control RNA,and at least one exogenous control RNA. In some embodiments, a method ofdetecting bladder cancer comprises detecting the levels of a set ofmarkers consisting of CRH, IGF2, KRT20 and ANXA10 mRNA, at least oneendogenous control RNA, and at least one exogenous control RNA.

In the present disclosure, the term “target RNA” is used for convenienceto refer to CRH, IGF2, KRT20 and ANXA10 mRNAs and also to other targetRNAs, such as exogenous and/or endogenous control RNAs. Thus, it is tobe understood that when a discussion is presented in terms of a targetRNA, that discussion is specifically intended to encompass CRH, IGF2,KRT20 and ANXA10 mRNAs, and/or other target RNAs.

In some embodiments, the level of one or more target RNAs is detected ina urine sample. In some embodiments, the level of one or more targetRNAs is determined in a urine sample that has been preserved in a mannerthat causes damage, such as lysis, to red blood cells and/or white bloodcells. In some embodiments, the level of one or more target RNAs isdetected in urothelial cells isolated from a urine sample, either withor without preservative treatment. In some embodiments, the urothelialcells are isolated by filtration.

In some embodiments, detection of an elevated level of one or moretarget RNAs selected from CRH, IGF2, KRT20 and ANXA10 in a sample from asubject indicates the presence of bladder cancer in the subject. In someembodiments, the detecting is done quantitatively. In other embodiments,the detecting is done qualitatively. In some embodiments, detecting atarget RNA comprises forming a complex comprising a polynucleotide and anucleic acid selected from a target RNA, a DNA amplicon of a target RNA,and a complement of a target RNA. In some embodiments, detecting atarget RNA comprises RT-PCR. In some embodiments, detecting a target RNAcomprises quantitative RT-PCT. In some embodiments, the level of thetarget RNA is compared to a normal or control level of the target RNA.

In some embodiments, the levels of target RNAs, such as CRH, IGF2, KRT20and ANXA10 mRNA, can be measured in samples collected at one or moretimes from a patient to monitor the status or progression of bladdercancer in the patient. In some embodiments, a patient with a history ofbladder cancer, such as a history of low grade bladder cancer or ahistory of high grade bladder cancer, is monitored by detecting thelevels of CRH, IGF2, KRT20 and ANXA10 mRNA at regular or semi-regularintervals. In some such embodiments, the patient is monitored bydetecting the levels of the target RNAs at least once per month, atleast once every two months, at least once every three months, at leastonce every four months, at least once every five months, at least onceevery six months, at least once every nine months, at least once peryear, or at least once every two years.

In some embodiments, a sample to be tested is a urine sample (such as avoided urine sample), or is derived from a urine sample. In someembodiments, a preservative is added to the urine sample, for example,to damage (e.g., lyse) red and/or white blood cells present in the urinesample. By damaging or lysing red and/or white blood cells prior toisolation of urothelial cells, contamination by the red and/or whiteblood cells can be reduced. In some embodiments, the urine sample iscentrifuged to concentrate the urothelial cells. In some embodiments,the urine sample is filtered to isolate the urothelial cells from otherurine and preservative materials. In some such embodiments, the filteris part of a GeneXpert cartridge (Cepheid, Sunnyvale, Calif.).

In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, orless than 2 ml of urine are used in the present methods. In someembodiments, the urine sample is analyzed without a centrifugation step.Thus, in some embodiments, the present methods are carried out in theabsence of centrifugation. In some embodiments, a larger volume of urinemay be used, and in some such embodiments, a centrifugation step may beused to concentrate the urothelial cells prior to analysis.

In some embodiments, the sample to be tested is another bodily fluid,such as blood, sputum, mucus, saliva, semen, etc. In some embodiments, asample to be tested is a blood sample. In some embodiments, the bloodsample is whole blood. In some embodiments, the blood sample is a sampleof blood cells. In some embodiments, the blood sample is plasma. In someembodiments, the blood sample is serum.

The clinical sample to be tested is, in some embodiments, fresh (i.e.,never frozen). In other embodiments, the sample is a frozen specimen. Insome embodiments, the sample is a tissue sample, such as aformalin-fixed paraffin embedded sample. In some embodiments, the sampleis a liquid cytology sample.

In some embodiments, the methods described herein are used for earlydetection of bladder cancer in a sample of urothelial cells, such asthose obtained from voided urine. In some embodiments, the methodsdescribed herein are used for monitoring for recurrence of bladdercancer using a sample of urothelial cells, such as those obtained fromvoided urine.

In some embodiments, the sample to be tested is obtained from anindividual who has one or more of the following risk factors: history ofsmoking, hematuria, history of bladder or other cancers, and exposure toknown carcinogens such as benzene. In some embodiments, the sample isobtained from an individual who has diagnostic signs or clinicalsymptoms that may be associated with bladder cancer, such as blood inthe urine, frequent urination, urinary urgency, incontinence, difficultyurinating, abdominal pain, unexplained weight loss and/or loss ofappetite. In some embodiments, the sample to be tested is obtained froman individual who has previously been diagnosed with low grade or highgrade bladder cancer. In some such embodiments, the individual ismonitored for recurrence of bladder cancer.

Bladder cancer can be divided into stages, which indicate the growthpattern of the primary tumor. Table A shows the stages of bladder canceraccording to the American Joint Committee on Cancer (AJCC). The stagesshown cover only the “T” portion of the “TNM” system. The “T” portionrefers to the primary tumor, while “N” refers to spread of the cancer tothe lymph nodes, and “M” refers to whether the cancer has metastasizedto distant sites.

TABLE A Bladder cancer stages Stage description T0 No evidence ofprimary tumor Ta Non-invasive papillary carcinoma Tis/ Non-invasive flatcarcinoma (carcinoma in situ) CIS T1 Tumor has grown from the lining ofthe bladder into the connective tissue, but has not grown into themuscle layer of the bladder T2 Tumor has grown into the muscle layer T2aTumor has grown into the inner half of the muscle layer T2b Tumor hasgrown into the outer half of the muscle layer T3 Tumor has grown throughthe muscle layer of the bladder and into the fatty tissue that surroundsit T3a Tumor's spread to fatty tissue can only be seen under amicroscope T3b Tumor's spread to fatty tissue can be seen on imagingtests or can be seen or felt by surgeon T4 Tumor has spread beyond thefatty tissue to nearby organs or structures T4a Tumor has spread to thestroma of the prostate, or to the uterus and/or vagina T4b Tumor hasspread to the pelvic wall or abdominal wall

In some embodiments, methods described herein can be used for routinescreening of healthy individuals with no risk factors. In someembodiments, methods described herein are used to screen asymptomaticindividuals having one or more of the above-described risk factors.

In some embodiments, the methods described herein can be used to detectlow grade bladder cancer. In some embodiments, the methods describedherein can be used to detect high grade bladder cancer. In someembodiments, the methods described herein detect at least 15%, at least17%, at least 20%, at least 22%, at least 25%, at least 27%, at least30%, at least 31%, at least 32%, at least 33%, at least 34%, or at least35% of low grade bladder cancers. In some embodiments, the methodsdescribed herein detect at least 85%, at least 87%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% of high gradebladder cancers.

In some embodiments, the methods described herein can be used to assessthe effectiveness of a treatment for bladder cancer in a patient. Insome embodiments, target RNA levels, such as the levels of CRH, IGF2,KRT20 or ANXA10 mRNA, are determined at various times during thetreatment, and are compared to target RNA levels from an archival sampletaken from the patient before the beginning of treatment. In someembodiments, target RNA levels are compared to target RNA levels from anarchival normal sample taken from the patient. Ideally, target RNAlevels in the normal sample evidence no aberrant changes in target RNAlevels.

In some embodiments, use of the levels of CRH, IGF2, KRT20 and ANXA10mRNA for monitoring recurrence of bladder cancer is provided. In someembodiments, an elevated level of one or more, two or more, three ormore, or all four mRNAs indicates that bladder cancer has recurred inthe patient.

In any of the embodiments described herein, RNA levels may be detectedconcurrently or simultaneously in the same or separate assay reactions.In some embodiments, RNA levels are detected at different times, e.g.,in serial assay reactions.

In some embodiments, a method comprises detecting the level of CRH,IGF2, KRT20 and ANXA10 mRNA in a sample from a subject, whereindetection of a level of any one of the four mRNAs that is greater than anormal level of the RNA indicates the presence of bladder cancer in thesubject. In some embodiments, detection of elevated levels of two,three, or four bladder cancer marker mRNAs indicates the presence ofbladder cancer in the subject. In some embodiments, detection ofelevated levels of at least two, at least three, or all four of thebladder cancer marker mRNAs indicates a greater risk of high gradebladder cancer.

In some embodiments, a method of facilitating diagnosis of bladdercancer in a subject is provided. Such methods comprise detecting thelevels of CRH, IGF2, KRT20 and ANXA10 mRNA in a sample from the subject.In some embodiments, information concerning the levels of CRH, IGF2,KRT20 and/or ANXA10 mRNA in the sample from the subject is communicatedto a medical practitioner. A “medical practitioner,” as used herein,refers to an individual or entity that diagnoses and/or treats patients,such as a hospital, a clinic, a physician's office, a physician, anurse, or an agent of any of the aforementioned entities andindividuals. In some embodiments, detecting the levels of CRH, IGF2,KRT20 and ANXA10 mRNA is carried out at a laboratory that has receivedthe subject's sample from the medical practitioner or agent of themedical practitioner. The laboratory carries out the detection by anymethod, including those described herein, and then communicates theresults to the medical practitioner. A result is “communicated,” as usedherein, when it is provided by any means to the medical practitioner. Insome embodiments, such communication may be oral or written, may be bytelephone, in person, by e-mail, by mail or other courier, or may bemade by directly depositing the information into, e.g., a databaseaccessible by the medical practitioner, including databases notcontrolled by the medical practitioner. In some embodiments, theinformation is maintained in electronic form. In some embodiments, theinformation can be stored in a memory or other computer readable medium,such as RAM, ROM, EEPROM, flash memory, computer chips, digital videodiscs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape,etc.

In some embodiments, methods of detecting the presence bladder cancerare provided. In some embodiments, methods of diagnosing bladder cancerare provided. In some embodiments, the method comprises obtaining asample from a subject and providing the sample to a laboratory fordetection of levels of CRH, IGF2, KRT20 and ANXA10 mRNA in the sample.In some embodiments, the method further comprises receiving acommunication from the laboratory that indicates the level of at leastone RNA selected from CRH, IGF2, KRT20 and ANXA10 mRNA in the sample. Insome embodiments, bladder cancer is present if the level of any one ofthe four mRNAs is greater than a normal or control level of the mRNA. A“laboratory,” as used herein, is any facility that detects the levels ofCRH, IGF2, KRT20 and ANXA10 mRNA in a sample by any method, includingthe methods described herein, and communicates the level to a medicalpractitioner. In some embodiments, a laboratory is under the control ofa medical practitioner. In some embodiments, a laboratory is not underthe control of the medical practitioner.

When a laboratory communicates the level of at least one RNA selectedfrom CRH, IGF2, KRT20 and ANXA10 to a medical practitioner, in someembodiments, the laboratory communicates a numerical value representingthe level of the RNA in the sample, with or without providing anumerical value for a normal level. In some embodiments, the laboratorycommunicates the level of the RNA by providing a qualitative value, suchas “high,” “low,” “elevated,” “decreased,” “positive” (such as “CRHpositive” or “CRH and KRT20 positive”), etc. In some embodiments, thelaboratory communicates a suggested diagnosis, such as “bladder cancerpositive” or “positive for cancer,” and the like; or simply “cancerpositive” or “cancer negative.”

As used herein, when a method relates to detecting bladder cancer,determining the presence of bladder cancer, monitoring for bladdercancer, and/or diagnosing bladder cancer, the method includes activitiesin which the steps of the method are carried out, but the result isnegative for the presence of bladder cancer. That is, detecting,determining, monitoring, and diagnosing bladder cancer include instancesof carrying out the methods that result in either positive or negativeresults.

In some embodiments, more than one RNA is detected simultaneously in asingle reaction. In some embodiments, CRH, IGF2, KRT20 and ANXA10 mRNAsare detected simultaneously in a single reaction. In some embodiments,CRH, IGF2, KRT20 and ANXA10 mRNAs and at least one endogenous controland/or at least one exogenous control are detected simultaneously in asingle reaction. In some embodiments, CRH, IGF2, KRT20 and ANXA10 mRNAs,and endogenous control, and an exogenous control are detectedsimultaneously in a single reaction.

6.2.2. Exemplary Controls

In some embodiments, a normal level (a “control”) of a target RNA, suchas CRH, IGF2, KRT20 or ANXA10 mRNA, can be determined as an averagelevel or range that is characteristic of normal urothelial cells orother reference material, against which the level measured in the samplecan be compared. The determined average or range of a target RNA innormal subjects can be used as a benchmark for detecting above-normallevels of the target RNA that are indicative of bladder cancer. In someembodiments, normal levels of a target RNA can be determined usingindividual or pooled RNA-containing samples from one or moreindividuals, such as from normal urothelial cells isolated from urine ofhealthy individuals.

In some embodiments, determining a normal level of a target RNA, such asCRH, IGF2, KRT20 or ANXA10 mRNA, comprises detecting a complexcomprising a polynucleotide for detection hybridized to a nucleic acidselected from a target RNA, a DNA amplicon of the target RNA, and acomplement of the target RNA. That is, in some embodiments, a normallevel can be determined by detecting a DNA amplicon of the target RNA,or a complement of the target RNA rather than the target RNA itself. Insome embodiments, a normal level of such a complex is determined andused as a control. The normal level of the complex, in some embodiments,correlates to the normal level of the target RNA. Thus, when a normallevel of a target is discussed herein, that level can, in someembodiments, be determined by detecting such a complex.

In some embodiments, a normal level of a target RNA is, or has been,determined by the same method as the level of the target RNA from apatient sample. In some such embodiments, the method is RT-PCR (such asreal-time RT-PCR, quantitative RT-PCR, etc.).

In some embodiments, a control comprises RNA from cells of a singleindividual, e.g., from normal urothelial cells isolated from urine of ahealthy individual. In some embodiments, a control comprises RNA fromblood, such as whole blood or serum, of a single individual. In someembodiments, a control comprises RNA from a pool of cells from multipleindividuals. In some embodiments, a control comprises RNA from a pool ofurine from multiple individuals. In some embodiments, a controlcomprises commercially-available human RNA (see, for example, Ambion).In some embodiments, a normal level or normal range has already beenpredetermined prior to testing a sample for an elevated level.

In some embodiments, the normal level of a target RNA can be determinedfrom one or more continuous cell lines, typically cell lines previouslyshown to have levels of RNAs that approximate the levels in normalurothelial cells.

In some embodiments, quantitation of target RNA levels requiresassumptions to be made about the total RNA per cell and the extent ofsample loss during sample preparation. In order to correct fordifferences between different samples or between samples that areprepared under different conditions, the quantities of target RNAs insome embodiments are normalized to the levels of at least one endogenouscontrol and/or at least one exogenous control.

In some embodiments, a control RNA is an endogenous control RNA. Anendogenous control RNA may be any RNA suitable for the purpose, forexample, RNAs that are present at approximately constant levels fromcell to cell and in urothelial cells from both bladder cancer andnon-bladder cancer patients. Nonlimiting exemplary endogenous controlRNAs include ABL, GUSB, GAPDH, TUBB, and UPK1a. In some embodiments, oneendogenous control is used for normalization. In some embodiments, morethan one endogenous control is used for normalization.

In some embodiments, the level of a target RNA, such as CRH, IGF2, KRT20or ANXA10 mRNA, is normalized to an endogenous control RNA.Normalization may comprise, for example, determination of the differenceof the level of the target RNA to the level of the endogenous controlRNA. In some such embodiments, the level of the RNAs are represented bya Ct value obtained from quantitative PCR. In some such embodiments, thedifference is expressed as ΔCt. ΔCt may be calculated as Ct[targetRNA]-Ct[endogenous control] or Ct[endogenous control]-Ct[target RNA]. Incertain embodiments, ΔCt=Ct[endogenous control]−Ct[marker]. In someembodiments, a threshold ΔCt value is set, above or below which bladdercancer is indicated. In some such embodiments, the ΔCt threshold is setas the ΔCt value below which 95% of normal samples are correctlycharacterized. In some such embodiments, a ΔCt value that is higher thanthe threshold ΔCt value is indicative of bladder cancer.

In some embodiments, linear discriminant analysis (LDA) is used, forexample, to combine two or more of the markers into a single combinedscale. In some such embodiments, a single threshold value is used forthe markers included in the LDA.

In some embodiments, a control RNA is an exogenous control RNA. In somesuch embodiments, the exogenous control RNA is an Armored RNA®, which isprotected by a bacteriophage coat. An exogenous control RNA may, in someembodiments, be used to determine if the detection assay reaction hasfailed, and therefore the results are not meaningful. For example, if anexogenous control RNA is not amplified in the assay reaction, then anegative result for the target RNAs is likely not meaningful because thelevels reflect the reaction failing rather than the target RNA levelsbeing low. Reaction failure can occur for any number of reasons,including, but not limited to, the presence of a reaction inhibitor inthe sample (an “inhibitory sample”), compromised reagents, the presenceof an RNAse, etc. An exogenous RNA control may be added at any stage ofthe sample collection and analysis. For example, in some embodiments,the exogenous control RNA is added to the sample at the timepreservative is added, is added to the sample when it is received by thediagnostic laboratory, is added to the sample immediately prior toanalysis, or is added to the sample during analysis (as a nonlimitingexample, during or after lysis of the urothelial cells but beforeaddition of the amplification reagents).

In some embodiments, the level of a target RNA, such as such as CRH,IGF2, KRT20 or ANXA10 mRNA, is compared to a reference level, e.g., froma confirmed bladder cancer. In some such embodiments, a similar level ofa target RNA relative to the reference sample indicates bladder cancer.

In some embodiments, a level of a target RNA, such as CRH, IGF2, KRT20or ANXA10 mRNA, that is at least about 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% greater than a normal level of the respective target RNAindicates the presence of bladder cancer. In some embodiments, a levelof a target RNA, such as CRH, IGF2, KRT20 or ANXA10 mRNA, that is atleast about two-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 6-fold, at least about 7-fold, atleast about 8-fold, at least about 9-fold, or at least about 10-foldgreater than a normal level of the respective target RNA indicates thepresence of bladder cancer.

In some embodiments, a control level of a target RNA, such as CRH, IGF2,KRT20 or ANXA10 mRNA, is determined contemporaneously, such as in thesame assay or batch of assays, as the level of the target RNA in asample. In some embodiments, a control level of a target RNA is notdetermined contemporaneously as the level of the target RNA in a sample.In some such embodiments, the control level has been determinedpreviously.

In some embodiments, the level of an endogenous control and/or anexogenous control is determined contemporaneously, such as in the sameassay or batch of assays, as the level of the target RNA in a sample. Insome embodiments, an assay comprises reagents for determining the levelsof CRH, IGF2, KRT20 and ANXA10 mRNA, and an endogenous controlsimultaneously in the same assay reaction. In some embodiments, an assaycomprises reagents for determining the levels of CRH, IGF2, KRT20 andANXA10 mRNA, and an exogenous control simultaneously in the same assayreaction. In some embodiments, an assay comprises reagents fordetermining the levels of CRH, IGF2, KRT20, ANXA10 mRNA, an endogenouscontrol, and an exogenous control simultaneously in the same assayreaction. In some such embodiments, for example, an assay reactioncomprises primer sets for amplifying each of CRH, IGF2, KRT20 and ANXA10mRNAs, a primer set for amplifying an endogenous control and/or a primerset for amplifying an exogenous control, and detectably differentlabeled probes for detecting the amplification products (such as, forexample, TaqMan® probes with detectably different dyes for eachdifferent amplicon to be detected).

In some embodiments, the level of a target RNA is not compared to acontrol level, for example, when it is known that the target RNA ispresent at very low levels, or not at all, in normal cells. In suchembodiments, detection of a high level of the target RNA in a sample isindicative of bladder cancer.

6.2.3. Exemplary Sample Preparation

6.2.3.1. Exemplary Urine Preservatives

In some embodiments, a preservative is added to the urine sample. Insome embodiments, the preservative is added within one hour, two hours,three hours, or six hours of the time the urine sample was collected(e.g., voided). In some embodiments, a preservative is added to theurine sample within one hour, two hours, three hours, or six hoursbefore the sample is analyzed by the methods described herein.

In some embodiments, a preservative causes damage, such as lysis, of redblood cells and/or white blood cells, but does not damage urothelialcells. Red blood cells and/or white blood cells may be present in theurine as a result of a tumor and/or infection. In some such embodiments,adding the preservative allows for improved enrichment of the urothelialcells, for example by filtration. In some embodiments, a preservativelowers the pH of the urine sample and improves solubility of urinesalts. In some such embodiments, the preservative facilitates passage ofthe salts through a filter in a filtration step. A desirable pH ofpreserved urine to be passed through a filter is between about 2.5 and4. In some embodiments, a desirable pH of preserved urine is betweenabout 2.7 and 3.7. In some embodiments, a desirable pH of preservedurine is between about 3 and 3.5. In some embodiments, a desirable pH ofpreserved urine is about 3.2.

In some embodiments a preservative is added such that theurine/preservative sample comprises 0.875M to 2.625M guanidinehydrochloride, 0.25% to 0.75% N-acetyl-L-cysteine, 6.25 to 18.75 mMsodium citrate, and 0.625% to 1.875% Tween-20, and has a pH of 3 to 3.5.In some embodiments a preservative is added such that theurine/preservative sample comprises about 1.75 M guanidinehydrochloride, about 0.5% N-acetyl-L-cysteine, about 12.5 mM sodiumcitrate, and about 1.25% Tween-20, and has a pH of about 3.2.

A nonlimiting exemplary commercial preservative is PreservCyt (Hologic,Bedford, Mass.).

6.2.3.2. Exemplary Cell Enrichment

In some embodiments, urothelial cells are enriched by centrifugation. Insome such embodiments, the cell pellet is resuspended in the supernatantand/or a preservative. Resuspension of the cell pellet can be used toadjust the concentration of cells in solution. The resuspended cellpellet may be used (for example, with lysis) in the methods describedherein, or may be subject to an additional enrichment step, such asfiltration.

In some embodiments, urothelial cells are enriched by filtration.Nonlimiting exemplary filter pore sizes that may be suitable forcapturing urothelial cells include 0.8 μm, 2 μm, 8 μm, and 10 μm. Insome embodiments, a filter pore size is selected that allowspass-through or red blood cells and/or white blood cells, whileretaining most urothelial cells. In some embodiments, a filter islocated within a GeneXpert cartridge designed for carrying out a bladdercancer diagnostic assay described herein.

6.2.3.3. Exemplary mRNA Preparation

Target RNA can be prepared by any appropriate method. Total RNA can beisolated by any method, including, but not limited to, the protocols setforth in Wilkinson, M. (1988) Nucl. Acids Res. 16(22):10,933; andWilkinson, M. (1988) Nucl. Acids Res. 16(22): 10934, or by usingcommercially-available kits or reagents, such as the TRIzol® reagent(Invitrogen), Total RNA Extraction Kit (iNtRON Biotechnology), Total RNAPurification Kit (Norgen Biotek Corp.), RNAqueous™ (Ambion), MagMAX™(Ambion), RecoverAll™ (Ambion), RNeasy (Qiagen), etc.

In some embodiments, RNA levels are measured in a sample in which RNAhas not first been purified from the cells. In some such embodiments,the cells are subject to a lysis step to release the RNA. Nonlimitingexemplary lysis methods include sonication (for example, for 2-15seconds, 8-18 μm at 36 kHz); chemical lysis, for example, using adetergent; and various commercially available lysis reagents (such asRNeasy lysis buffer, Qiagen). In some embodiments, RNA levels aremeasured in a sample in which RNA has been isolated.

In some embodiments, RNA is modified before a target RNA, such as CRH,IGF2, KRT20 or ANXA10 mRNA, is detected. In some embodiments, all of theRNA in the sample is modified. In some embodiments, just the particulartarget RNAs to be analyzed are modified, e.g., in a sequence-specificmanner. In some embodiments, RNA is reverse transcribed. In some suchembodiments, RNA is reverse transcribed using MMLV reversetranscriptase. Nonlimiting exemplary conditions for reverse transcribingRNA using MMLV reverse transcriptase include incubation from 5 to 20minutes at 40° C. to 50° C.

When a target RNA is reverse transcribed, a DNA complement of the targetRNA is formed. In some embodiments, the complement of a target RNA isdetected rather than a target RNA itself (or a DNA copy of the RNAitself). Thus, when the methods discussed herein indicate that a targetRNA is detected, or the level of a target RNA is determined, suchdetection or determination may be carried out on a complement of atarget RNA instead of, or in addition to, the target RNA itself. In someembodiments, when the complement of a target RNA is detected rather thanthe target RNA, a polynucleotide for detection is used that iscomplementary to the complement of the target RNA. In some suchembodiments, a polynucleotide for detection comprises at least a portionthat is identical in sequence to the target RNA, although it may containthymidine in place of uridine, and/or comprise other modifiednucleotides.

6.2.4. Exemplary Analytical Methods

As described above, methods are presented for detecting bladder cancer.The methods comprise detecting a panel of bladder cancer markersconsisting of CRH, IGF2, KRT20 and ANXA10, and optionally including atleast one endogenous control and/or at least one exogenous control. Insome embodiments, detection of an elevated level of one of the fourbladder cancer markers indicates the presence of bladder cancer. In someembodiments, detection of an elevated level of two, three, or all fourof the bladder cancer markers indicates the presence of bladder cancer.In some embodiments, the bladder cancer is low grade bladder cancer. Insome embodiments, the bladder cancer is high grade bladder cancer. Insome embodiments, the bladder cancer is a recurrence of bladder cancerin a patient with a history of bladder cancer.

Any analytical procedure capable of permitting specific and quantifiable(or semi-quantifiable) detection of a target RNA, such as CRH, IGF2,KRT20 and ANXA10 mRNAs, may be used in the methods herein presented.Such analytical procedures include, but are not limited to, RT-PCRmethods, and other methods known to those skilled in the art.

In some embodiments, the method of detecting a target RNA, such as CRH,IGF2, KRT20 or ANXA10 mRNA, comprises amplifying cDNA complementary tothe target RNA. Such amplification can be accomplished by any method.Exemplary methods include, but are not limited to, real time PCR,endpoint PCR, and amplification using T7 polymerase from a T7 promoterannealed to a cDNA, such as provided by the SenseAmp Plus™ Kit availableat Implen, Germany.

When a target RNA or a cDNA complementary to a target RNA is amplified,in some embodiments, a DNA amplicon of the target RNA is formed. A DNAamplicon may be single stranded or double-stranded. In some embodiments,when a DNA amplicon is single-stranded, the sequence of the DNA ampliconis related to the target RNA in either the sense or antisenseorientation. In some embodiments, a DNA amplicon of a target RNA isdetected rather than the target RNA itself. Thus, when the methodsdiscussed herein indicate that a target RNA is detected, or the level ofa target RNA is determined, such detection or determination may becarried out on a DNA amplicon of the target RNA instead of, or inaddition to, the target RNA itself. In some embodiments, when the DNAamplicon of the target RNA is detected rather than the target RNA, apolynucleotide for detection is used that is complementary to thecomplement of the target RNA. In some embodiments, when the DNA ampliconof the target RNA is detected rather than the target RNA, apolynucleotide for detection is used that is complementary to the targetRNA. Further, in some embodiments, multiple polynucleotides fordetection may be used, and some polynucleotides may be complementary tothe target RNA and some polynucleotides may be complementary to thecomplement of the target RNA.

In some embodiments, the method of detecting one or more targetRNAs—such as CRH, IGF2, KRT20 or ANXA10 mRNA, comprises RT-PCR, asdescribed below. In some embodiments, detecting one or more target RNAscomprises real-time monitoring of an RT-PCR reaction, which can beaccomplished by any method. Such methods include, but are not limitedto, the use of TaqMan®, Molecular beacon, or Scorpion probes (i.e.,energy transfer (ET) probes, such as FRET probes) and the use ofintercalating dyes, such as SYBR green, EvaGreen, thiazole orange,YO-PRO, TO-PRO, etc.

Nonlimiting exemplary conditions for amplifying cDNA that has beenreverse transcribed from the target RNAs are as follows. An exemplarycycle comprises an initial denaturation at 90° C. to 100° C. for 2 to 5minutes, followed by cycling that comprises denaturation at 90° C. to100° C. for 1 to 10 seconds, annealing at 60° C. to 70° C. for 10 to 30seconds, and extension at 60° C. to 75° C. for 10 to 40 seconds. In someembodiments, for the first cycle following the initial denaturationstep, the cycle denaturation step is omitted. In some embodiments, Taqpolymerase is used for amplification. In some embodiments, the cycle iscarried out at least 10 times, at least 15 times, at least 20 times, atleast 25 times, at least 30 times, at least 35 times, or at least 45times. In some such embodiments, Taq is used with a hot start function.In some embodiments, the amplification reaction occurs in a GeneXpertcartridge, and amplification of the four bladder cancer marker targetRNAs occurs in the same reaction. In some embodiments, detection of CRH,IGF2, KRT20 and ANXA10 mRNAs occurs in less than 3 hours, less than 2.5hours, or less than 2 hours, from initial denaturation through the lastextension.

In some embodiments, detection of a target RNA comprises forming acomplex comprising a polynucleotide that is complementary to a targetRNA or to a complement thereof, and a nucleic acid selected from thetarget RNA, a DNA amplicon of the target RNA, and a complement of thetarget RNA. Thus, in some embodiments, the polynucleotide forms acomplex with a target RNA. In some embodiments, the polynucleotide formsa complex with a complement of the target RNA, such as a cDNA that hasbeen reverse transcribed from the target RNA. In some embodiments, thepolynucleotide forms a complex with a DNA amplicon of the target RNA.When a double-stranded DNA amplicon is part of a complex, as usedherein, the complex may comprise one or both strands of the DNAamplicon. Thus, in some embodiments, a complex comprises only one strandof the DNA amplicon. In some embodiments, a complex is a triplex andcomprises the polynucleotide and both strands of the DNA amplicon. Insome embodiments, the complex is formed by hybridization between thepolynucleotide and the target RNA, complement of the target RNA, or DNAamplicon of the target RNA. The polynucleotide, in some embodiments, isa primer or probe.

In some embodiments, a method comprises detecting the complex. In someembodiments, the complex does not have to be associated at the time ofdetection. That is, in some embodiments, a complex is formed, thecomplex is then dissociated or destroyed in some manner, and componentsfrom the complex are detected. An example of such a system is a TaqMan®assay. In some embodiments, when the polynucleotide is a primer,detection of the complex may comprise amplification of the target RNA, acomplement of the target RNA, or a DNA amplicon of a target RNA.

In some embodiments the analytical method used for detecting at leastone target RNA in the methods set forth herein includes real-timequantitative RT-PCR. In some embodiments, the analytical method used fordetecting at least one target RNA includes the use of a TaqMan® probe.The assay uses energy transfer (“ET”), such as fluorescence resonanceenergy transfer (“FRET”), to detect and quantitate the synthesized PCRproduct. Typically, the TaqMan® probe comprises a fluorescent dyemolecule coupled to the 5′-end and a quencher molecule coupled to the3′-end, such that the dye and the quencher are in close proximity,allowing the quencher to suppress the fluorescence signal of the dye viaFRET. When the polymerase replicates the chimeric amplicon template towhich the TaqMan® probe is bound, the 5′-nuclease of the polymerasecleaves the probe, decoupling the dye and the quencher so that the dyesignal (such as fluorescence) is detected. Signal (such as fluorescence)increases with each RT-PCR cycle proportionally to the amount of probethat is cleaved.

In some embodiments, quantitation of the results of real-time RT-PCRassays is done by constructing a standard curve from a nucleic acid ofknown concentration and then extrapolating quantitative information fortarget RNAs of unknown concentration. In some embodiments, the nucleicacid used for generating a standard curve is an RNA (for example, anendogenous control, or an exogenous control). In some embodiments, thenucleic acid used for generating a standard curve is a purifieddouble-stranded plasmid DNA or a single-stranded DNA generated in vitro.

In some embodiments, where the amplification efficiencies of the targetnucleic acids and the endogenous reference are approximately equal,quantitation is accomplished by the comparative Ct (cycle threshold,e.g., the number of PCR cycles required for the fluorescence signal torise above background) method. Ct values are inversely proportional tothe amount of nucleic acid target in a sample. In some embodiments, Ctvalues of a target RNA can be compared with a control or calibrator,such an exogenous control RNA. In some embodiments, the Ct values of theexogenous control and the target RNA are normalized to an appropriateendogenous control. Nonlimiting exemplary endogenous controls arediscussed herein.

In some embodiments, a threshold Ct (or a “cutoff Ct”) value for atarget RNA, below which bladder cancer is indicated, has previously beendetermined. In such embodiments, a control sample may not be assayedconcurrently with the test sample. In some embodiments, as discussedherein, a ΔCt threshold value is determined, above which bladder canceris indicated, has previously been determined.

In addition to the TaqMan® assays, other real-time RT-PCR chemistriesuseful for detecting and quantitating PCR products in the methodspresented herein include, but are not limited to, Molecular Beacons,Scorpion probes and intercalating dyes, such as SYBR Green, EvaGreen,thiazole orange, YO-PRO, TO-PRO, etc., which are discussed below.

In various embodiments, real-time RT-PCR detection is utilized todetect, in a single multiplex reaction, all four bladder cancer markersof the panel described herein, and optionally, at least one endogenouscontrol and/or at least one exogenous control. In some multiplexembodiments, a plurality of probes, such as TaqMan® probes, eachspecific for a different RNA target, is used. In some embodiments, eachtarget RNA-specific probe is spectrally distinguishable from the otherprobes used in the same multiplex reaction.

In some embodiments, quantitation of real-time RT PCR products isaccomplished using a dye that binds to double-stranded DNA products,such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc. Insome embodiments, the assay is the QuantiTect SYBR Green PCR assay fromQiagen. In this assay, total RNA is first isolated from a sample. TotalRNA is subsequently poly-adenylated at the 3′-end and reversetranscribed using a universal primer with poly-dT at the 5′-end. In someembodiments, a single reverse transcription reaction is sufficient toassay multiple target RNAs. Real-time RT-PCR is then accomplished usingtarget RNA-specific primers and an miScript Universal Primer, whichcomprises a poly-dT sequence at the 5′-end. SYBR Green dye bindsnon-specifically to double-stranded DNA and upon excitation, emitslight. In some embodiments, buffer conditions that promotehighly-specific annealing of primers to the PCR template (e.g.,available in the QuantiTect SYBR Green PCR Kit from Qiagen) can be usedto avoid the formation of non-specific DNA duplexes and primer dimersthat will bind SYBR Green and negatively affect quantitation. Thus, asPCR product accumulates, the signal from SYBR Green increases, allowingquantitation of specific products.

Real-time RT-PCR is performed using any RT-PCR instrumentation availablein the art. Typically, instrumentation used in real-time RT-PCR datacollection and analysis comprises a thermal cycler, optics forfluorescence excitation and emission collection, and optionally acomputer and data acquisition and analysis software.

In some embodiments, the analytical method used in the methods describedherein is a DASL® (cDNA-mediated Annealing, Selection, Extension, andLigation) Assay. In some embodiments, total RNA is isolated from asample to be analyzed by any method. Total RNA may then bepolyadenylated (>18 A residues are added to the 3′-ends of the RNAs inthe reaction mixture). The RNA is reverse transcribed using abiotin-labeled DNA primer that comprises from the 5′ to the 3′ end, asequence that includes a PCR primer site and a poly-dT region that bindsto the poly-dA tail of the sample RNA. The resulting biotinylated cDNAtranscripts are then hybridized to a solid support via abiotin-streptavidin interaction and contacted with one or more targetRNA-specific polynucleotides. The target RNA-specific polynucleotidescomprise, from the 5′-end to the 3′-end, a region comprising a PCRprimer site, region comprising an address sequence, and a targetRNA-specific sequence.

In some DASL® embodiments, the target RNA-specific sequence comprises atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19 contiguous nucleotides having a sequence that is the same as,or complementary to, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19 contiguous nucleotides of a bladdercancer marker target RNA, an endogenous control RNA, or an exogenouscontrol RNA.

After hybridization, the target RNA-specific polynucleotide is extended,and the extended products are then eluted from the immobilized cDNAarray. A second PCR reaction using a fluorescently-labeled universalprimer generates a fluorescently-labeled DNA comprising the targetRNA-specific sequence. The labeled PCR products are then hybridized to amicrobead array for detection and quantitation.

In some embodiments, the analytical method used for detecting andquantifying the levels of the at least one target RNA in the methodsdescribed herein is a bead-based flow cytometric assay. See Lu J. et al.(2005) Nature 435:834-838, which is incorporated herein by reference inits entirety. An example of a bead-based flow cytometric assay is thexMAP® technology of Luminex, Inc. (Seehttp://www.luminexcorp.com/technology/index.html). In some embodiments,total RNA is isolated from a sample and is then labeled with biotin. Thelabeled RNA is then hybridized to target RNA-specific capture probes(e.g., FlexmiR™ products sold by Luminex, Inc. athttp://www.luminexcorp.com/products/assays/index.html) that arecovalently bound to microbeads, each of which is labeled with 2 dyeshaving different fluorescence intensities. A streptavidin-bound reportermolecule (e.g., streptavidin-phycoerythrin, also known as “SAPE”) isattached to the captured target RNA and the unique signal of each beadis read using flow cytometry. In some embodiments, the RNA sample isfirst polyadenylated, and is subsequently labeled with a biotinylated3DNA™ dendrimer (i.e., a multiple-arm DNA with numerous biotin moleculesbound thereto), using a bridging polynucleotide that is complementary tothe 3′-end of the poly-dA tail of the sample RNA and to the 5′-end ofthe polynucleotide attached to the biotinylated dendrimer. Thestreptavidin-bound reporter molecule is then attached to thebiotinylated dendrimer before analysis by flow cytometry. In someembodiments, biotin-labeled RNA is first exposed to SAPE, and theRNA/SAPE complex is subsequently exposed to an anti-phycoerythrinantibody attached to a DNA dendrimer, which can be bound to as many as900 biotin molecules. This allows multiple SAPE molecules to bind to thebiotinylated dendrimer through the biotin-streptavidin interaction, thusincreasing the signal from the assay.

In some embodiments, the analytical method used for detecting andquantifying the levels of the at least one target RNA in the methodsdescribed herein is by gel electrophoresis and detection with labeledprobes (e.g., probes labeled with a radioactive or chemiluminescentlabel), such as by Northern blotting. In some embodiments, total RNA isisolated from the sample, and then is size-separated by SDSpolyacrylamide gel electrophoresis. The separated RNA is then blottedonto a membrane and hybridized to radiolabeled complementary probes. Insome embodiments, exemplary probes contain one or moreaffinity-enhancing nucleotide analogs as discussed below, such as lockednucleic acid (“LNA”) analogs, which contain a bicyclic sugar moietyinstead of deoxyribose or ribose sugars. See, e.g., Varallyay, E. et al.(2008) Nature Protocols 3(2):190-196, which is incorporated herein byreference in its entirety.

In some embodiments, detection and quantification of one or more targetRNAs is accomplished using microfluidic devices and single-moleculedetection. In some embodiments, target RNAs in a sample of isolatedtotal RNA are hybridized to two probes, one which is complementary tonucleic acids at the 5′-end of the target RNA and the second which iscomplementary to the 3′-end of the target RNA. Each probe comprises, insome embodiments, one or more affinity-enhancing nucleotide analogs,such as LNA nucleotide analogs and each is labeled with a differentfluorescent dye having different fluorescence emission spectra (i.e.,detectably different dyes). The sample is then flowed through amicrofluidic capillary in which multiple lasers excite the fluorescentprobes, such that a unique coincident burst of photons identifies aparticular target RNA, and the number of particular unique coincidentbursts of photons can be counted to quantify the amount of the targetRNA in the sample. In some alternative embodiments, a targetRNA-specific probe can be labeled with 3 or more distinct labelsselected from, e.g., fluorophores, electron spin labels, etc., and thenhybridized to an RNA sample.

Optionally, the sample RNA is modified before hybridization. The targetRNA/probe duplex is then passed through channels in a microfluidicdevice and that comprise detectors that record the unique signal of the3 labels. In this way, individual molecules are detected by their uniquesignal and counted. See U.S. Pat. Nos. 7,402,422 and 7,351,538 to Fuchset al., U.S. Genomics, Inc., each of which is incorporated herein byreference in its entirety.

6.2.5. Exemplary Automation and Systems

In some embodiments, gene expression is detected using an automatedsample handling and/or analysis platform. In some embodiments,commercially available automated analysis platforms are utilized. Forexample, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale,Calif.) is utilized.

The present invention is illustrated for use with the GeneXpert system.Exemplary sample preparation and analysis methods are described below.However, the present invention is not limited to a particular detectionmethod or analysis platform. One of skill in the art recognizes that anynumber of platforms and methods may be utilized.

The GeneXpert® utilizes a self-contained, single use cartridge. Sampleextraction, amplification, and detection may all carried out within thisself-contained “laboratory in a cartridge.” (See e.g., U.S. Pat. Nos.5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which isherein incorporated by reference in its entirety.)

Components of the cartridge include, but are not limited to, processingchambers containing reagents, filters, and capture technologies usefulto extract, purify, and amplify target nucleic acids. A valve enablesfluid transfer from chamber to chamber and contain nucleic acids lysisand filtration components. An optical window enables real-time opticaldetection. A reaction tube enables very rapid thermal cycling.

In some embodiments, the GenXpert® system includes a plurality ofmodules for scalability. Each module includes a plurality of cartridges,along with sample handling and analysis components.

In some embodiments, the GeneXpert® sample preparation method utilizesfiltration in order to capture and concentrate cells from urine. In someembodiments, a filter pore size of 0.8 μm is utilized. This sizefacilitates capture of all cells in urine. In other embodiments, poresizes of 0.5 to 10 μm, 0.5 to 5 μm, 0.8 to 10 μm, 0.8 to 5 μm, 0.8 to 2μm, 2 to 5 μm, 2 to 10 μm, 2 to 8 μm, 5 to 8 μm, or 5 to 10 μm areutilized. Certain filters (such as 5 μm, 8 μm, and 10 μm) allow theremoval of most red and white blood cells from the sample whilecapturing the larger urothelial cells, which are the assay target cells.In some embodiments, this sample preparation method improves assayspecificity by removing white blood cells that may be present due toinfection or inflammation. In some instances, sample preparation methodssuch as centrifugation of whole urine followed by RNA isolation from theurine pellet do not allow for removal of white blood cells. In someembodiments, the efficiency of cell capture by filtration is highercompared to centrifugation, and may provide more consistent results.

After the cells from the urine are captured on the filter, in someembodiments, they are washed and then lysed using sonication (2-15seconds, 8-16 μm at 36 kHz). The cell lysate is then collected and usedto reconstitute the RT-PCR reagents, which are present in the cartridgeas lyophilized particles.

In some embodiments, RT-PCR is used to amplify and analyze the presenceor expression levels of the bladder cancer markers. In some embodiments,the reverse transcription uses MMLV RT enzyme and an incubation of 5 to20 minutes at 40° C. to 50° C. In some embodiments, the PCR uses Taqpolymerase with hot start function, such as AptaTaq (Roche). In someembodiments, the initial denaturation is at 90° C. to 100° C. for 2 to 5minutes; the cycling denaturation temperature is 90° C. to 100° C. for 1to 10 seconds; the cycling anneal temperature is 60° C. to 70° C. for 10to 30 seconds; and the cycling extend temperature is 60° C. to 75° C.for 10 to 40 seconds; and up to 50 cycles are performed.

The present invention is not limited to particular primer and/or probesequences. Exemplary amplification primers and detection probes aredescribed in the Examples.

In some embodiments, an off-line centrifugation is used to improve assayresults with samples with low cellular content. The sample, with orwithout the preservative added, is centrifuged and the supernatantremoved. The pellet is then resuspended in a smaller volume of eithersupernatant or the preservative. The resuspended pellet is then added toa GeneXpert® cartridge as previously described.

6.2.6. Exemplary Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., theexpression level of the bladder cancer markers described herein) intodata of predictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some embodiments, thepresent invention provides the further benefit that the clinician, whois not likely to be trained in genetics or molecular biology, need notunderstand the raw data. The data is presented directly to the clinicianin its most useful form. The clinician is then able to immediatelyutilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., expression level of the bladdercancer markers described herein or diagnosis of bladder cancer) for thesubject, along with recommendations for particular treatment options.The data may be displayed to the clinician by any suitable method. Forexample, in some embodiments, the profiling service generates a reportthat can be printed for the clinician (e.g., at the point of care) ordisplayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease or as acompanion diagnostic to determine a treatment course of action.

6.2.7. Exemplary Polynucleotides

In some embodiments, polynucleotides are provided. In some embodiments,synthetic polynucleotides are provided. Synthetic polynucleotides, asused herein, refer to polynucleotides that have been synthesized invitro either chemically or enzymatically. Chemical synthesis ofpolynucleotides includes, but is not limited to, synthesis usingpolynucleotide synthesizers, such as OligoPilot (GE Healthcare), ABI3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymaticsynthesis includes, but is not limited, to producing polynucleotides byenzymatic amplification, e.g., PCR. A polynucleotide may comprise one ormore nucleotide analogs (i.e., modified nucleotides) discussed herein.

In some embodiments, a polynucleotide is provided that comprises aregion that is identical to, or complementary to, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of a sequence selected from CRH, IGF2, KRT20, andANXA10 mRNA. In some embodiments, a polynucleotide is provided thatcomprises a region that is identical to, or complementary to, a span of6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 contiguousnucleotides of a sequence selected from CRH, IGF2, KRT20, and ANXA10mRNA. Nonlimiting exemplary polynucleotides are shown in Tables 1 and 6.

In various embodiments, a polynucleotide comprises fewer than 500, fewerthan 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75,fewer than 50, fewer than 40, or fewer than 30 nucleotides. In variousembodiments, a polynucleotide is between 6 and 200, between 8 and 200,between 8 and 150, between 8 and 100, between 8 and 75, between 8 and50, between 8 and 40, or between 8 and 30 nucleotides long.

In some embodiments, the polynucleotide is a primer. In someembodiments, the primer is labeled with a detectable moiety. In someembodiments, a primer is not labeled. A primer, as used herein, is apolynucleotide that is capable of specifically hybridizing to a targetRNA or to a cDNA reverse transcribed from the target RNA or to anamplicon that has been amplified from a target RNA or a cDNA(collectively referred to as “template”), and, in the presence of thetemplate, a polymerase and suitable buffers and reagents, can beextended to form a primer extension product.

In some embodiments, the polynucleotide is a probe. In some embodiments,the probe is labeled with a detectable moiety. A detectable moiety, asused herein, includes both directly detectable moieties, such asfluorescent dyes, and indirectly detectable moieties, such as members ofbinding pairs. When the detectable moiety is a member of a binding pair,in some embodiments, the probe can be detectable by incubating the probewith a detectable label bound to the second member of the binding pair.In some embodiments, a probe is not labeled, such as when a probe is acapture probe, e.g., on a microarray or bead. In some embodiments, aprobe is not extendable, e.g., by a polymerase. In other embodiments, aprobe is extendable.

In some embodiments, the polynucleotide is a FRET probe that in someembodiments is labeled at the 5′-end with a fluorescent dye (donor) andat the 3′-end with a quencher (acceptor), a chemical group that absorbs(i.e., suppresses) fluorescence emission from the dye when the groupsare in close proximity (i.e., attached to the same probe). In otherembodiments, the dye and quencher are not at the ends of the FRET probe.Thus, in some embodiments, the emission spectrum of the dye shouldoverlap considerably with the absorption spectrum of the quencher.

6.2.7.1. Exemplary Polynucleotide Modifications

In some embodiments, the methods of detecting at least one target RNAdescribed herein employ one or more polynucleotides that have beenmodified, such as polynucleotides comprising one or moreaffinity-enhancing nucleotide analogs. Modified polynucleotides usefulin the methods described herein include primers for reversetranscription, PCR amplification primers, and probes. In someembodiments, the incorporation of affinity-enhancing nucleotidesincreases the binding affinity and specificity of a polynucleotide forits target nucleic acid as compared to polynucleotides that contain onlydeoxyribonucleotides, and allows for the use of shorter polynucleotidesor for shorter regions of complementarity between the polynucleotide andthe target nucleic acid.

In some embodiments, affinity-enhancing nucleotide analogs includenucleotides comprising one or more base modifications, sugarmodifications and/or backbone modifications.

In some embodiments, modified bases for use in affinity-enhancingnucleotide analogs include 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthineand hypoxanthine.

In some embodiments, affinity-enhancing nucleotide analogs includenucleotides having modified sugars such as 2′-substituted sugars, suchas 2′-O-alkyl-ribose sugars, 2′-amino-deoxyribose sugars,2′-fluoro-deoxyribose sugars, 2′-fluoro-arabinose sugars, and2′-O-methoxyethyl-ribose (2′MOE) sugars. In some embodiments, modifiedsugars are arabinose sugars, or d-arabino-hexitol sugars.

In some embodiments, affinity-enhancing nucleotide analogs includebackbone modifications such as the use of peptide nucleic acids (PNA;e.g., an oligomer including nucleobases linked together by an amino acidbackbone). Other backbone modifications include phosphorothioatelinkages, phosphodiester modified nucleic acids, combinations ofphosphodiester and phosphorothioate nucleic acid, methylphosphonate,alkylphosphonates, phosphate esters, alkylphosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters, methylphosphorothioate,phosphorodithioate, p-ethoxy, and combinations thereof.

In some embodiments, a polynucleotide includes at least oneaffinity-enhancing nucleotide analog that has a modified base, at leastnucleotide (which may be the same nucleotide) that has a modified sugar,and/or at least one internucleotide linkage that is non-naturallyoccurring.

In some embodiments, an affinity-enhancing nucleotide analog contains alocked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In someembodiments, a polynucleotide for use in the methods described hereincomprises one or more nucleotides having an LNA sugar. In someembodiments, a polynucleotide contains one or more regions consisting ofnucleotides with LNA sugars. In other embodiments, a polynucleotidecontains nucleotides with LNA sugars interspersed withdeoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm.Des. 14(11):1138-1142.

6.2.7.2. Exemplary Primers

In some embodiments, a primer is provided. In some embodiments, a primeris identical to, or complementary to, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, or at least 30 contiguousnucleotides of a sequence selected from CRH, IGF2, KRT20, and ANXA10mRNA. In some embodiments, a primer is provided that comprises a regionthat is identical to, or complementary to, a span of 6 to 100, 8 to 100,8 to 75, 8 to 50, 8 to 40, or 8 to 30 contiguous nucleotides of asequence selected from CRH, IGF2, KRT20, and ANXA10 mRNA. Nonlimitingexemplary primers are shown in Tables 1 and 6. In some embodiments, aprimer may also comprise portions or regions that are not identical orcomplementary to the target RNA. In some embodiments, a region of aprimer that is identical or complementary to a target RNA is contiguous,such that any region of a primer that is not identical or complementaryto the target RNA does not disrupt the identical or complementaryregion.

In some embodiments, a primer comprises a portion that is identicallypresent in a target RNA. In some such embodiments, a primer thatcomprises a region that is identically present in the target RNA iscapable of selectively hybridizing to a cDNA that has been reversetranscribed from the RNA, or to an amplicon that has been produced byamplification of the target RNA or cDNA. In some embodiments, the primeris complementary to a sufficient portion of the cDNA or amplicon suchthat it selectively hybridizes to the cDNA or amplicon under theconditions of the particular assay being used.

As used herein, “selectively hybridize” means that a polynucleotide,such as a primer or probe, will hybridize to a particular nucleic acidin a sample with at least 5-fold greater affinity than it will hybridizeto another nucleic acid present in the same sample that has a differentnucleotide sequence in the hybridizing region. Exemplary hybridizationconditions are discussed herein, for example, in the context of areverse transcription reaction or a PCR amplification reaction. In someembodiments, a polynucleotide will hybridize to a particular nucleicacid in a sample with at least 10-fold greater affinity than it willhybridize to another nucleic acid present in the same sample that has adifferent nucleotide sequence in the hybridizing region.

In some embodiments, a primer is used to reverse transcribe a targetRNA, for example, as discussed herein. In some embodiments, a primer isused to amplify a target RNA or a cDNA reverse transcribed therefrom.Such amplification, in some embodiments, is quantitative PCR, forexample, as discussed herein. In some embodiments, a primer comprises adetectable moiety.

In some embodiments, primer pairs are provided. Such primer pairs aredesigned to amplify a portion of a target mRNA, such as CRH, IGF2,KRT20, or ANXA10 mRNA, or an endogenous control RNA, or an exogenouscontrol RNA. In some embodiments, a primer pair is designed to producean amplicon that is 50 to 1500 nucleotides long, 50 to 1000 nucleotideslong, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotideslong, 50 to 150 nucleotides long, or 50 to 100 nucleotides long.Nonlimiting exemplary primer pairs are shown in Tables 1 and 6. In someembodiments, a primer pair is designed that spans an intron in thegenomic sequence so that the mRNA, without the intron, is morepreferably amplified than the genomic sequence. By “spans an intron” ismeant that one primer of the primer pair is complementary to a sequencein the mRNA or a cDNA reverse transcribed from the mRNA that is at leastpartially located 5′ to an intron in the genomic sequence and one primerof the primer pair is complementary to a sequence in the mRNA or a cDNAreverse transcribed from the mRNA that is at least partially located 3′to the same intron in the genomic sequence. In some embodiments, oneprimer of the primer pair is complementary to a sequence in the mRNA ora cDNA reverse transcribed from the mRNA that is located 5′ to an intronin the genomic sequence and one primer of the primer pair iscomplementary to a sequence in the mRNA or a cDNA reverse transcribedfrom the mRNA that is located 3′ to the same intron in the genomicsequence. In some embodiments, one of the primers in the primer pair maybe complementary to a sequence in the mRNA or a cDNA reverse transcribedfrom the mRNA that is spliced together when the intron is removed suchthat the contiguous complementary sequence is not found in the genomicsequence. A primer pair comprising such a primer is still considered tospan an intron.

6.2.7.3. Exemplary Probes

In various embodiments, methods of detecting the presence of bladdercancer comprise hybridizing nucleic acids of a sample with a probe. Insome embodiments, the probe comprises a portion that is complementary toa target RNA, such as CRH, IGF2, KRT20 or ANXA10 mRNA. In someembodiments, the probe comprises a portion that is identically presentin the target RNA. In some such embodiments, a probe that iscomplementary to a target RNA is complementary to a sufficient portionof the target RNA such that it selectively hybridizes to the target RNAunder the conditions of the particular assay being used. In someembodiments, a probe that is complementary to a target RNA comprises aregion that is complementary to at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, or at least 30 contiguous nucleotides ofthe target RNA, such as CRH, IGF2, KRT20 or ANXA10 mRNA. Nonlimitingexemplary probes are shown in Tables 1 and 6. A probe that iscomplementary to a target RNA may also comprise portions or regions thatare not complementary to the target RNA. In some embodiments, a regionof a probe that is complementary to a target RNA is contiguous, suchthat any region of a probe that is not complementary to the target RNAdoes not disrupt the complementary region.

In some embodiments, the probe comprises a portion that is identicallypresent in the target RNA, such as CRH, IGF2, KRT20 or ANXA10 mRNA. Insome such embodiments, a probe that comprises a region that isidentically present in the target RNA is capable of selectivelyhybridizing to a cDNA that has been reverse transcribed from the RNA, orto an amplicon that has been produced by amplification of the target RNAor cDNA. In some embodiments, the probe is complementary to a sufficientportion of the cDNA or amplicon such that it selectively hybridizes tothe cDNA or amplicon under the conditions of the particular assay beingused. In some embodiments, a probe that is complementary to a cDNA oramplicon comprises a region that is complementary to at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 26, at least 27, at least 28, at least 29, or at least 30contiguous nucleotides of the cDNA or amplicon. A probe that iscomplementary to a cDNA or amplicon may also comprise portions orregions that are not complementary to the cDNA or amplicon. In someembodiments, a region of a probe that is complementary to a cDNA oramplicon is contiguous, such that any region of a probe that is notcomplementary to the cDNA or amplicon does not disrupt the complementaryregion.

In some embodiments, the method of detectably quantifying one or moretarget RNAs comprises: (a) reverse transcribing a target RNA to producea cDNA that is complementary to the target RNA; (b) amplifying the cDNAfrom (a); and (c) detecting the amount of a target RNA using real timeRT-PCR and a detection probe (which may be simultaneous with theamplification step (b)).

As described above, in some embodiments, real time RT-PCR detection maybe performed using a FRET probe, which includes, but is not limited to,a TaqMan® probe, a Molecular beacon probe and a Scorpion probe. In someembodiments, the real time RT-PCR detection and quantification isperformed with a TaqMan® probe, i.e., a linear probe that typically hasa fluorescent dye covalently bound at one end of the DNA and a quenchermolecule covalently bound at the other end of the DNA. The FRET probecomprises a sequence that is complementary to a region of the cDNA suchthat, when the FRET probe is hybridized to the cDNA, the dyefluorescence is quenched, and when the probe is digested duringamplification of the cDNA, the dye is released from the probe andproduces a fluorescence signal. In such embodiments, the amount oftarget RNA in the sample is proportional to the amount of fluorescencemeasured during cDNA amplification.

The TaqMan′ probe typically comprises a region of contiguous nucleotideshaving a sequence that is complementary to a region of a target RNA orits complementary cDNA that is reverse transcribed from the target RNAtemplate (i.e., the sequence of the probe region is complementary to oridentically present in the target RNA to be detected) such that theprobe is specifically hybridizable to the resulting PCR amplicon. Insome embodiments, the probe comprises a region of at least 6 contiguousnucleotides having a sequence that is fully complementary to oridentically present in a region of a cDNA that has been reversetranscribed from a target RNA template, such as comprising a region ofat least 8 contiguous nucleotides, at least 10 contiguous nucleotides,at least 12 contiguous nucleotides, at least 14 contiguous nucleotides,or at least 16 contiguous nucleotides having a sequence that iscomplementary to or identically present in a region of a cDNA reversetranscribed from a target RNA to be detected.

In some embodiments, the region of the cDNA that has a sequence that iscomplementary to the TaqMan® probe sequence is at or near the center ofthe cDNA molecule. In some embodiments, there are independently at least2 nucleotides, such as at least 3 nucleotides, such as at least 4nucleotides, such as at least 5 nucleotides of the cDNA at the 5′-endand at the 3′-end of the region of complementarity.

In some embodiments, Molecular Beacons can be used to detect andquantitate PCR products. Like TaqMan® probes, Molecular Beacons use FRETto detect and quantitate a PCR product via a probe having a fluorescentdye and a quencher attached at the ends of the probe. Unlike TaqMan®probes, Molecular Beacons remain intact during the PCR cycles. MolecularBeacon probes form a stem-loop structure when free in solution, therebyallowing the dye and quencher to be in close enough proximity to causefluorescence quenching. When the Molecular Beacon hybridizes to atarget, the stem-loop structure is abolished so that the dye and thequencher become separated in space and the dye fluoresces. MolecularBeacons are available, e.g., from Gene Link™ (seehttp://www.genelink.com/newsite/products/mbintro.asp).

In some embodiments, Scorpion probes can be used as bothsequence-specific primers and for PCR product detection andquantitation. Like Molecular Beacons, Scorpion probes form a stem-loopstructure when not hybridized to a target nucleic acid. However, unlikeMolecular Beacons, a Scorpion probe achieves both sequence-specificpriming and PCR product detection. A fluorescent dye molecule isattached to the 5′-end of the Scorpion probe, and a quencher is attachedto the 3′-end. The 3′ portion of the probe is complementary to theextension product of the PCR primer, and this complementary portion islinked to the 5′-end of the probe by a non-amplifiable moiety. After theScorpion primer is extended, the target-specific sequence of the probebinds to its complement within the extended amplicon, thus opening upthe stem-loop structure and allowing the dye on the 5′-end to fluoresceand generate a signal. Scorpion probes are available from, e.g, PremierBiosoft International (see http://www.premierbiosoft.com/technotes/Scorpion.html).

In some embodiments, labels that can be used on the FRET probes includecolorimetric and fluorescent dyes such as Alexa Fluor dyes, BODIPY dyes,such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and itsderivatives, such as 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins anderythrosins; fluorescein and its derivatives, such as fluoresceinisothiocyanate; macrocyclic chelates of lanthanide ions, such as QuantumDye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red,tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energytransfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.

Specific examples of dyes include, but are not limited to, thoseidentified above and the following: Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and,BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE,Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG,Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2′,4′,5′,7′-Tetrabromosulfonefluorescein, and TET.

Examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, butare not limited to, fluorescein/tetramethylrhodamine;IAEDANS/fluorescein; EDANS/dabcyl; fluorescein/fluorescein; BODIPYFL/BODIPY FL; fluorescein/QSY 7 or QSY 9 dyes. When the donor andacceptor are the same, FRET may be detected, in some embodiments, byfluorescence depolarization. Certain specific examples of dye/quencherpairs (i.e., donor/acceptor pairs) include, but are not limited to,Alexa Fluor 350/Alexa Fluor488; Alexa Fluor 488/Alexa Fluor 546; AlexaFluor 488/Alexa Fluor 555; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor488/Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647; Alexa Fluor546/Alexa Fluor 568; Alexa Fluor 546/Alexa Fluor 594; Alexa Fluor546/Alexa Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor555/Alexa Fluor 647; Alexa Fluor 568/Alexa Fluor 647; Alexa Fluor594/Alexa Fluor 647; Alexa Fluor 350/QSY35; Alexa Fluor 350/dabcyl;Alexa Fluor 488/QSY 35; Alexa Fluor 488/dabcyl; Alexa Fluor 488/QSY 7 orQSY 9; Alexa Fluor 555/QSY 7 or QSY9; Alexa Fluor 568/QSY 7 or QSY 9;Alexa Fluor 568/QSY 21; Alexa Fluor 594/QSY 21; and Alexa Fluor 647/QSY21. In some instances, the same quencher may be used for multiple dyes,for example, a broad spectrum quencher, such as an Iowa Black® quencher(Integrated DNA Technologies, Coralville, Iowa) or a Black HoleQuencher™ (BHQ™; Sigma-Aldrich, St. Louis, Mo.).

In some embodiments, for example, in a multiplex reaction in which twoor more moieties (such as amplicons) are detected simultaneously, eachprobe comprises a detectably different dye such that the dyes may bedistinguished when detected simultaneously in the same reaction. Oneskilled in the art can select a set of detectably different dyes for usein a multiplex reaction.

Specific examples of fluorescently labeled ribonucleotides useful in thepreparation of RT-PCR probes for use in some embodiments of the methodsdescribed herein are available from Molecular Probes (Invitrogen), andthese include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPYFL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescentribonucleotides are available from Amersham Biosciences (GE Healthcare),such as Cy3-UTP and Cy5-UTP.

Examples of fluorescently labeled deoxyribonucleotides useful in thepreparation of RT-PCR probes for use in the methods described hereininclude Dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPYFL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPYTMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, AlexaFluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPYTR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commerciallyavailable and can be purchased from, e.g., Invitrogen.

In some embodiments, dyes and other moieties, such as quenchers, areintroduced into polynucleotide used in the methods described herein,such as FRET probes, via modified nucleotides. A “modified nucleotide”refers to a nucleotide that has been chemically modified, but stillfunctions as a nucleotide. In some embodiments, the modified nucleotidehas a chemical moiety, such as a dye or quencher, covalently attached,and can be introduced into a polynucleotide, for example, by way ofsolid phase synthesis of the polynucleotide. In other embodiments, themodified nucleotide includes one or more reactive groups that can reactwith a dye or quencher before, during, or after incorporation of themodified nucleotide into the nucleic acid. In specific embodiments, themodified nucleotide is an amine-modified nucleotide, i.e., a nucleotidethat has been modified to have a reactive amine group. In someembodiments, the modified nucleotide comprises a modified base moiety,such as uridine, adenosine, guanosine, and/or cytosine. In specificembodiments, the amine-modified nucleotide is selected from5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP;N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments,nucleotides with different nucleobase moieties are similarly modified,for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Manyamine modified nucleotides are commercially available from, e.g.,Applied Biosystems, Sigma, Jena Bioscience and TriLink.

Exemplary detectable moieties also include, but are not limited to,members of binding pairs. In some such embodiments, a first member of abinding pair is linked to a polynucleotide. The second member of thebinding pair is linked to a detectable label, such as a fluorescentlabel. When the polynucleotide linked to the first member of the bindingpair is incubated with the second member of the binding pair linked tothe detectable label, the first and second members of the binding pairassociate and the polynucleotide can be detected. Exemplary bindingpairs include, but are not limited to, biotin and streptavidin,antibodies and antigens, etc.

In some embodiments, multiple target RNAs are detected in a singlemultiplex reaction. In some such embodiments, each probe that istargeted to a unique cDNA is spectrally distinguishable when releasedfrom the probe. Thus, each target RNA is detected by a uniquefluorescence signal.

One skilled in the art can select a suitable detection method for aselected assay, e.g., a real-time RT-PCR assay. The selected detectionmethod need not be a method described above, and may be any method.

6.3. Exemplary Compositions and Kits

In another aspect, compositions are provided. In some embodiments,compositions are provided for use in the methods described herein.

In some embodiments, compositions are provided that comprise at leastone target RNA-specific primer. The term “target RNA-specific primer”encompasses primers that have a region of contiguous nucleotides havinga sequence that is (i) identically present in a target RNA, such as CRH,IGF2, KRT20, or ANXA10 mRNA, or (ii) complementary to the sequence of aregion of contiguous nucleotides found in a target RNA, such CRH, IGF2,KRT20, or ANXA10 mRNA. In some embodiments, a composition is providedthat comprises at least one pair of target RNA-specific primers. Theterm “pair of target RNA-specific primers” encompasses pairs of primersthat are suitable for amplifying a defined region of a target RNA, suchas CRH, IGF2, KRT20, or ANXA10 mRNA. A pair of target RNA-specificprimers typically comprises a first primer that comprises a sequencethat is identical to the sequence of a region of a target RNA (althoughthe primer will typically comprise DNA or modified nucleosides ratherthan RNA) and a second primer that comprises a sequence that iscomplementary to a region of a target RNA. A pair of primers istypically suitable for amplifying a region of a target mRNA that is 50to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotideslong, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 tO 150nucleotides long, or 50 to 100 nucleotides long. Nonlimiting exemplaryprimers, and pairs of primers, are shown in Tables 1 and 6.

In some embodiments, a composition comprises four pairs of targetRNA-specific primers, one pair for amplifying each of CRH, IGF2, KRT20,and ANXA10 mRNA. In some embodiments, a composition additionallycomprises a pair of target RNA-specific primers for amplifying anendogenous control RNA and/or one pair of target RNA-specific primersfor amplifying an exogenous control RNA.

In some embodiments, a composition comprises at least one targetRNA-specific probe. The term “target RNA-specific probe” encompassesprobes that have a region of contiguous nucleotides having a sequencethat is (i) identically present in a target RNA, such as such as CRH,IGF2, KRT20, or ANXA10 mRNA, or (ii) complementary to the sequence of aregion of contiguous nucleotides found in a target RNA, such as such asCRH, IGF2, KRT20, or ANXA10 mRNA. Nonlimiting exemplary target-specificprobes are shown in Tables 1 and 6.

In some embodiments, a composition (including a composition describedabove that comprises four or more pairs of target RNA-specific primers)comprises four probes, one probe for detecting each of CRH, IGF2, KRT20,and ANXA10 mRNA. In some embodiments, a composition additionallycomprises a probe for detecting an endogenous control RNA and/or a probefor detecting an exogenous control RNA.

In some embodiments, a composition is an aqueous composition. In someembodiments, the aqueous composition comprises a buffering component,such as phosphate, tris, HEPES, etc., and/or additional components, asdiscussed below. In some embodiments, a composition is dry, for example,lyophilized, and suitable for reconstitution by addition of fluid. A drycomposition may include one or more buffering components and/oradditional components.

In some embodiments, a composition further comprises one or moreadditional components. Additional components include, but are notlimited to, salts, such as NaCl, KCl, and MgCl₂; polymerases, includingthermostable polymerases such as Taq; dNTPs; reverse transcriptases,such as MMLV reverse transcriptase; RNase inhibitors; bovine serumalbumin (BSA) and the like; reducing agents, such as β-mercaptoethanol;EDTA and the like; etc. One skilled in the art can select suitablecomposition components depending on the intended use of the composition.

In some embodiments, compositions are provided that comprise at leastone polynucleotide for detecting at least one target RNA. In someembodiments, the polynucleotide is used as a primer for a reversetranscriptase reaction. In some embodiments, the polynucleotide is usedas a primer for amplification. In some embodiments, the polynucleotideis used as a primer for RT-PCR. In some embodiments, the polynucleotideis used as a probe for detecting at least one target RNA. In someembodiments, the polynucleotide is detectably labeled. In someembodiments, the polynucleotide is a FRET probe. In some embodiments,the polynucleotide is a TaqMan® probe, a Molecular Beacon, or a Scorpionprobe.

In some embodiments, a composition comprises at least one FRET probehaving a sequence that is identically present in, or complementary to aregion of, CRH, IGF2, KRT20, or ANXA10 mRNA. In some embodiments, a FRETprobe is labeled with a donor/acceptor pair such that when the probe isdigested during the PCR reaction, it produces a unique fluorescenceemission that is associated with a specific target RNA. In someembodiments, when a composition comprises multiple FRET probes, eachprobe is labeled with a different donor/acceptor pair such that when theprobe is digested during the PCR reaction, each one produces a uniquefluorescence emission that is associated with a specific probe sequenceand/or target RNA. In some embodiments, the sequence of the FRET probeis complementary to a target region of a target RNA. In otherembodiments, the FRET probe has a sequence that comprises one or morebase mismatches when compared to the sequence of the best-aligned targetregion of a target RNA.

In some embodiments, a composition comprises a FRET probe consisting ofat least 8, at least 9, at least 10, at least 11, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, or atleast 25 nucleotides, wherein at least a portion of the sequence isidentically present in, or complementary to a region of, CRH, IGF2,KRT20, or ANXA10 mRNA. In some embodiments, at least 8, at least 9, atleast 10, at least 11, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 nucleotides of theFRET probe are identically present in, or complementary to a region of,CRH, IGF2, KRT20, or ANXA10 mRNA. In some embodiments, the FRET probehas a sequence with one, two or three base mismatches when compared tothe sequence or complement of small CRH, IGF2, KRT20, or ANXA10 mRNA.

In some embodiments, a kit comprises a polynucleotide discussed above.In some embodiments, a kit comprises at least one primer and/or probediscussed above. In some embodiments, a kit comprises at least onepolymerase, such as a thermostable polymerase. In some embodiments, akit comprises dNTPs. In some embodiments, kits for use in the real timeRT-PCR methods described herein comprise one or more target RNA-specificFRET probes and/or one or more primers for reverse transcription oftarget RNAs and/or one or more primers for amplification of target RNAsor cDNAs reverse transcribed therefrom.

In some embodiments, one or more of the primers and/or probes is“linear”. A “linear” primer refers to a polynucleotide that is a singlestranded molecule, and typically does not comprise a short region of,for example, at least 3, 4 or 5 contiguous nucleotides, which arecomplementary to another region within the same polynucleotide such thatthe primer forms an internal duplex. In some embodiments, the primersfor use in reverse transcription comprise a region of at least 4, suchas at least 5, such as at least 6, such as at least 7 or more contiguousnucleotides at the 3′-end that has a sequence that is complementary toregion of at least 4, such as at least 5, such as at least 6, such as atleast 7 or more contiguous nucleotides at the 5′-end of a target RNA.

In some embodiments, a kit comprises one or more pairs of linear primers(a “forward primer” and a “reverse primer”) for amplification of a cDNAreverse transcribed from a target RNA, such as CRH, IGF2, KRT20, orANXA10 mRNA. Accordingly, in some embodiments, a first primer comprisesa region of at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, or at least 25 contiguous nucleotides having a sequencethat is identical to the sequence of a region of at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25contiguous nucleotides at a first location in the mRNA. Furthermore, insome embodiments, a second primer comprises a region of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, or atleast 25 contiguous nucleotides having a sequence that is complementaryto the sequence of a region of at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, or at least 25 contiguousnucleotides at a second location in the mRNA, such that a PCR reactionusing the two primers results in an amplicon extending from the firstlocation of the mRNA to the second location of the mRNA.

In some embodiments, the kit comprises at least two, at least three, orat least four sets of primers, each of which is for amplification of acDNA that is reverse transcribed from a different target RNA, includingCRH, IGF2, KRT20, and ANXA10 mRNA. In some embodiments, the kit furthercomprises at least one set of primers for amplifying a control RNA, suchas an endogenous control and/or an exogenous control.

In some embodiments, probes and/or primers for use in the compositionsdescribed herein comprise deoxyribonucleotides. In some embodiments,probes and/or primers for use in the compositions described hereincomprise deoxyribonucleotides and one or more nucleotide analogs, suchas LNA analogs or other duplex-stabilizing nucleotide analogs describedabove. In some embodiments, probes and/or primers for use in thecompositions described herein comprise all nucleotide analogs. In someembodiments, the probes and/or primers comprise one or moreduplex-stabilizing nucleotide analogs, such as LNA analogs, in theregion of complementarity.

In some embodiments, the kits for use in real time RT-PCR methodsdescribed herein further comprise reagents for use in the reversetranscription and amplification reactions. In some embodiments, the kitscomprise enzymes such as reverse transcriptase, and a heat stable DNApolymerase, such as Taq polymerase. In some embodiments, the kitsfurther comprise deoxyribonucleotide triphosphates (dNTP) for use inreverse transcription and amplification. In further embodiments, thekits comprise buffers optimized for specific hybridization of the probesand primers.

The following examples are for illustration purposes only, and are notmeant to be limiting in any way.

7. EXAMPLES 7.1. Example 1: Detection of High Grade and Low GradeBladder Cancer

More than 30 mRNA markers and 20 microRNA markers were evaluated in bothbladder tissue and urine samples to determine the most accurate panelfor detection of bladder cancer. Based on results from over 200 urinesamples using eight markers and the GeneXpert system (Cepheid,Sunnyvale, Calif.), a panel consisting of CRH, IGF2, KRT20, and ANXA10mRNA markers (with or without at least one endogenous and/or at leastone exogenous control) was selected.

Various reaction compositions were designed for use in the GeneXpert®system, for detecting various combinations of CRH, IGF2, KRT20, andANXA10 mRNA. Table 1 shows the sequences of the primers and probes usedto detect each of the target RNAs by quantitative RT-PCR in the variousreaction compositions.

TABLE 1 Primer and probe sequences SEQ Reagent Formul. oligo name targetsequence ID NO (“TSR”) ABLa3a4 ABL GATCAACACTGCTTCTGATGGCAA  8CL3, CL4, CL1 PrmrFwd4 ABLa3a4 ABL CCACCGTTGAATGATGATGAACCAA  9CL3, CL4, CL1 PrmrRev1 ABLa3a4 Probe 1 ABL F4-CCTCCGAGAGCCGCTTCAAC-Q4 10CL3 ABL probe F6 ABL F6-CCTCCGAGAGCCGCTTCAAC-Q6 11 CL4 ABL probe F1 ABLF1-CCTCCGAGAGCCGC(T-dabsyl)TCAAC-Q1 12 CL1 KRT20 For KRT20TTGAAGAGCTGCGAAGTCAGAT 13 CL3, CL4 KRT20 Rev KRT20 TGAAGTCCTCAGCAGCCAGTT14 CL3, CL4 KRT20 Probe (F3) KRT20 F3-TCAACTGCAAAATGCTCGGTGTGTCC-Q3 15CL3, CL4 IGF2 For_4 IGF2 CGCGGCTTCTACTTCAGCAG 16 CL3, CL4 IGF2 Rev_4IGF2 GCGGAAACAGCACTCCTCAA 17 CL3, CL4 IGF2 Probe_2 IGF2F5-TGTGAGCCGTCGCAGCCGTG-Q5 18 CL3, CL4 CRH_For CRH ACCCGGCTCACCTGCGAA 19CL3, CL4, CL1 CRH_Rev CRH GGACTCCCGCGGACACAA 20 CL3, CL4, CL1CRH_probe 3 CRH F2-TCCTGGGAAGCGAGTGCCCCTAA-Q2 21 CL3, CL1 CRH_probe_F1CRH F1-CCTGGGAAGCGAG(T-Dabsyl)GCCCCTAA-Q1 22 CL4 Armored RNA ® exogenousTGCTTGAGCTCCAGTCCCTAAG 24 CL1 control Armored exogenousF6-AGCCGAGAAGGCGGAGTCTGGC-Q6 25 CL1 RNA ®_Probe control ANXA10-FW ANXA10GTGAAACAAGTTTATGCAATCGATCAA 26 CL1 ANXA10-RV3 ANXA10GATTGAAATTGGGAGCTGGGAA 27 CL1 ANXA10-F3 ANXA10F3-TCATCCCTGAGGTTAACAATTACCATCAA-Q3 28 CL1 F1 through F6 are detectablydifferent dyes that can be detected and distinguished simultaneously ina multiplex reaction, and Q1 to Q6 are quenchers (in the presentexample, Q2, Q4, Q5, and Q6 are the same quencher).

The final primer and probe compositions of three different reactioncompositions are shown in Table 2.

TABLE 2 Primers and probes in TSR CL3, CL4, and CL1 Final conc. Finalconc. Final conc. Target Label Purpose forw. primer rev primer probe TSRCL3 ABL F4 Normalization 400 nM 400 nM 150 nM (endogenous control) KRT20F3 Bladder cancer marker 400 nM 400 nM  75 nM IGF2 F5 Bladder cancermarker 400 nM 400 nM 200 nM CRH F2 Bladder cancer marker 400 nM 400 nM200 nM TSR CL4 ABL F6 Normalization 400 nM 400 nM 400 nM (endogenouscontrol) KRT20 F3 Bladder cancer marker 400 nM 400 nM  75 nM IGF2 F5Bladder cancer marker 400 nM 400 nM 300 nM CRH F1 Bladder cancer marker400 nM 400 nM 600 nM TSR CL1 ABL F1 Normalization 400 nM 400 nM 600 nM(endogenous control) Armored F6 Exogenous control 400 nM 400 nM 400 nMRNA ® ANXA10 F3 Bladder cancer marker 400 nM 400 nM  75 nM CRH F2Bladder cancer marker 400 nM 400 nM 300 nM

Each reaction contained 50-90 mM KCl, 3-5 mM MgCl₂, 400-825 μM dNTPs, 20mM Tris, pH 8.5, 0.01% sodium azide, and 0.9 units/μl of RNaseinhibitor. MMLV reverse transcriptase (0.375 units/μl) and AptaTaq (0.25units/μl; Roche) were used for reverse transcription and amplification,respectively. TSR CL1 included an Armored RNA® exogenous control (SEQ IDNO: 47; Asuragen, Austin, Tex.).

For each sample to be tested, 5 mL of voided urine was added to 5 mLpreservative (3.5M guanidine HCl, 1% N-acetyl-L-cysteine, 25 mM sodiumcitrate, and 2.5% Tween-20, pH 3.2), preferably within 1 hour of samplecollection. The preserved samples were transported on ice and stored at4° C. Clinical information for each sample was provided by thecollection sites. The number of red blood cells per millilitre wasdetermined by microscopic evaluation.

Prior to use, the preserved urine was inverted three times to mix. 1.2mL of preserved urine was loaded into a GeneXpert cartridge foranalysis. The cartridge contained a 0.8 μm filter to capture urothelialcells. The captured cells were washed and lysed using sonication (2-15seconds, 8-16 μm at 36 kHz) within the cartridge. The lysate was thenused to reconstitute the reagents used for real-time RT-PCR (describedabove). The reaction cycle used was: 10 minutes at 45° C., followed by 2minutes at 95° C., and then 45 cycles of (a) 5 seconds at 95° C., 20seconds at 60° C., and 20 seconds at 72° C., using a GeneXpert®cartridge in a GeneXpert® system. Delta Ct (ΔCt) was calculated as Ct(ABL)−Ct (marker). The ΔCt cutoff was set as the ΔCt that gave at least95% specificity with samples from patients not expected to have bladdercancer (data not shown). A ΔCt above the ΔCt cutoff for any one of themarkers was considered a positive result, indicative of the presence ofbladder cancer.

Some samples were also tested using UroVysion® (Abbott Laboratories,Abbott Park, Ill.). The results of that experiment are shown in Table 3(high grade bladder cancer) and Table 4 (low grade bladder cancer). ΔCtsabove the threshold, indicating a positive result, are highlighted. Eachof the three TSR lots, CL3, CL4, and CL1, detected 100% of high gradebladder cancer samples, as did UroVysion®.

For low grade bladder cancer, the detection rate was 37% (7/19),compared to only 16% (3/19) for UroVysion®.

TABLE 3 Detection of high grade bladder cancer history CIC TSR of TSRlot CL3 TSR lot CL4 lot CL1 Sample UroVysion ® bladder CRH KRT20 IGF2KRT20 IGF2 CRH ANXA10 CRH GX ID stage grade Result cytology cancer −102.2 −1 4 0.5 −5 −0.5 −3 result 67001 pTa high positive suspicious yes−20 4.9 −20 4.8 1.1 −20 −1.6 −3.6 positive 67006 pT1 high positivepositive no −0.7 4.4 −6.2 5.7 −2.8 −0.6 −20 2.2 positive 67009 pT2 highpositive positive no −20 4.9 −0.4 4.9 1.5 −20 1.1 −20 positive 75211 pT2high positive negative no −0.8 1.4 −4.1 positive 75216 pTa, highpositive suspicious yes −3.5 2 −0.8 0.9 −0.6 −3.4 positive CIC 75218 pTahigh positive negative no 0.9 3.8 −0.9 3.8 −1.6 0.5 positive 75245 pTahigh positive NA no 20 1.6 6.7 0.4 6.2 20 1.9 8.2 positive 75247 CIShigh positive atypical no −1.4 3.4 2.7 3.6 3.7 −3.8 −0.2 0.4 positive75248 pT1/ high positive atypical no −20 4.3 0 4.8 1 −20 0.4 −5.1positive CIS 75249 pT1 high positive negative no −20 3.3 2.1 3.4 2.5 −203.7 −20 positive 75258 CIS, high positive atypical no 4.2 −3.7 0.6 −21.9 positive pTa 75246 positive positive no −20 4.3 5.3 4.4 5.9 9.1 −13.4 positive

TABLE 4 Detection of low grade bladder cancer history CIC TSR of TSR lotCL3 TSR lot CL4 lot CL1 Sample UroVysion ® bladder CRH KRT20 IGF2 KRT20IGF2 CRH ANXA10 CRH GX ID stage grade Result cytology cancer −10 2.2 −14 0.5 −5 −0.5 −3 result 67002 pTa low negative atypical yes −20 3.9 −3.83.3 −1.5 −20 −1.7 −20 positive 67003 pTa low negative negative no −20−20 −3.2 −20 −20 −20 −20 −20 negative 67004 pTa low negative negative no−2 4.7 −2.3 5.2 0.1 −1.7 2.4 2.2 positive 67010 pTa low positiveatypical no −20 −5.3 −8.8 −3.8 −6.7 −20 −5 −20 negative 67011 pTa lowpositive atypical no −1.3 4.6 1.9 4 2.4 −2 −1.3 1 positive 67018 pTa lownegative atypical yes −20 −20 −4.4 −20 −20 −20 −20 −20 negative 67050pTa low negative atypical yes −20 −0.4 3.4 1.9 3.3 −20 −0.5 −20 positive67100 pTa low borderline atypical yes −2.9 −2.7 −20 −20 −20 negative75161 pTa low negative atypical yes −20 −3 −4.7 −3.6 −1.3 −20 −5.7 −20negative 75183 pTa low positive negative yes −1.5 3.1 −2 3.4 −0.5 0.7−4.1 2.3 positive 75184 pTa low inconclusive negative yes −20 −0.6 1.3−1.5 1.8 −20 −20 −20 positive 75185 pTa low negative negative no −20 3 51.3 4.7 −20 1.2 −4.2 positive 75191 pTa low negative negative yes −20−0.4 −3.4 1.1 −0.5 −20 −4.1 −20 negative 75202 pTa low negative negativeyes −20 0.3 −2.3 −0.4 −1.5 −20 −2.4 −20 negative 75236 pTa low negativenegative no −20 −5.7 −7.9 −20 −8.9 −20 −3.2 −20 negative 75251 pTa lownegative negative yes −20 −3.6 −5.1 −1 −4.4 −20 −20 −20 negative 75257pTa low negative negative no −2.1 −0.9 −20 −20 −20 negative 75265 pTalow negative negative yes 1.4 −20 −20 −20 −20 negative

A summary of the sensitivity for high grade bladder cancer and low gradebladder cancer, and the specificity in patients with a low risk ofbladder cancer, is shown in Table 5 for each of the individual markerstested in Tables 3 and 4.

TABLE 5 Summary of sensitivity and specificity of individual markersSensitivity, Sensitivity, Specificity, high grade low grade low risk ofMarker bladder cancer bladder cancer bladder cancer CRH 15/30 (50%) 9/53(17%) 220/221 (99%) KRT20 11/21 (52%) 6/34 (18%) 144/145 (99%) IGF213/21 (62%) 8/34 (24%) 144/145 (99%) ANXA10  5/9 (56%) 2/19 (11%)  74/76(97%) 4 marker combo 12/12 (100%)  7/19 (37%)  83/88 (94%)

Exemplary alternative primers and probes for detecting the four markers,KRT20, IGF2, CRH, and ANXA10, are shown in Table 6. Table 6 also showsan exemplary set of primers and probes for detecting an exogenouscontrol and an endogenous control, ABL. The dyes and quenchers shown inTable 6 are generic, and two or more of quenchers Q1 to Q6 may be thesame. One skilled in the art could select a suitable set, for example, aset of detectably different dyes for use in a multiplex assay. Thepredicted amplicon length for each set of primers is also shown, as wellas the length of any intervening intron(s) between the primer sites onthe genomic copy of the target.

TABLE 6 Primer and probe sequences amplicon intron SEQ length lengthname 5′ mod sequence 3′ mod ID NO (bp) (bp) KRT20 For_3CGACTACAGTGCATATTACAGACAA 29 113 2142 KRT20 Rev_3CAGCAGCCAGTTTAGCATTATCAA 30 KRT20 Probe F1 TCAACTGCAAAA(T-dabsyl)GCT Q131 CGGTGTGTCC IGF2 For_5 GGACCGCGGCTTCTACTTCA 32  95 1701 IGF2 Rev_5CCAGGTCACAGCTGCGGAA 33 IGF2 Probe_2_F4 F4 TGTGAGCCGTCGCAGCCGTG Q4 34CRH_For 4 TGCGAAGCGCCTGGGAAGC 35  66  801 CRH_Rev GGACTCCCGCGGACACAA 36CRH_probe_F2 F2 TGCCCCTAACATGCGGCTGCC Q2 37 ANXA10_For_3TCAGCGCTGCAATGCACAA 38 122 22, 947 ANXA10_For_4 CTGCAATGCACAAAGGATGA 48117 22, 947 ANXA10_Rev_5 GGCCAGCCATCACATCTTTGAA 39 ANXA10_Probe_3 F3TAGAGCATGTATGGCCGGGACCT Q3 40 ABLa3a4 GATCAACACTGCTTCTGATGGCAA 41  927666 PrmrFwd4 ABLa3a4 CCACCGTTGAATGATGATGAACCAA 42 PrmrRev1 ABL Probe F5F5 CCTCCGAGAGCCGCTTCAAC Q5 43 Armored RNA ® GGCTATTCTCCTCTTGGCAGAT 44101 NA Fwd Armored RNA ® TGCTTGAGCTCCAGTCCCTAAG 45 Rev Armored RNA ® F6AGCCGAGAAGGCGGAGTCTGGC Q6 46 Probe

7.2. Example 2: Assay Sensitivity for Detecting Bladder Cancer UsingMarker Panel KRT20, IGF2, CRH, and ANXA10 in a Larger Cohort

Urine samples were collected from subjects at seven different sites.Eligibility criteria for inclusion in the study included:

-   -   18 years or older;    -   Documented informed consent as required by the reviewing IRB or        HREC, and a signed Experimental Subjects Bill of Rights for        patients in California;    -   At least one of the following criteria:        -   A history or recurrence of bladder cancer;        -   A referral for cystoscopy evaluation due to micro- or            gross-hematuria in urine;        -   A referral for urology evaluation, but no previous history            of bladder cancer or clinical evidence of bladder cancer;    -   Consent to provide at least 15 ml voided urine in addition to        that required for standard of care;    -   Consent to allow pathology results for any biopsy specimens        taken during cystoscopy procedure and other medical records to        be reported.

Exclusion criteria included only under 18 years of age and first voidedurine. Patients currently or previously treated with BacillusCalmette-Guerin (BCG) and patients currently or previously treated withintravesical therapy or transurethral resection of bladder or radiationtherapy for bladder cancer were eligible for the study. In addition,repeat enrollment during the course of the study was also permitted.

Two of the collection sites provided the results of UroVysion® analysison the urine samples. For each sample to be tested 15 mL of voided urinewas added to 15 mL of preservative (3.5M guanidine HCl, 1%N-acetyl-L-cysteine, 25 mM sodium citrate, and 2.5% Tween-20, pH 3.2),preferably within 1 hour of sample collection. The preserved sampleswere transported on ice and stored at 4° C. Clinical information foreach sample was provided by the collection sites.

Prior to use, the preserved urine was inverted three times to mix. 4 mLof preserved urine was loaded into a GeneXpert cartridge for analysis.The cartridge contained a 0.8 μm filter to capture urothelial cells. Thecaptured cells were washed and lysed using sonication (2-15 seconds,8-16 μm at 36 kHz) within the cartridge. The lysate was then used toreconstitute the reagents used for real-time RT-PCR (described above).The reaction cycle used was: 10 minutes at 45° C., followed by 2 minutesat 95° C., and then 45 cycles of (a) 5 seconds at 95° C., 20 seconds at60° C., and 20 seconds at 72° C., using a GeneXpert® cartridge in aGeneXpert® system. For ANXA10, KRT20 and IGF2, delta Ct (ΔCt) wascalculated as Ct (ABL)−Ct (marker). The ΔCt cutoff was set as the ΔCtthat gave high (>90%) specificity with samples from patients notexpected to have bladder cancer (data not shown). A ΔCt above the ΔCtcutoff for any one of the markers was considered a positive result,indicative of the presence of bladder cancer. For CRH, Ct values wereused instead of ΔCt to determine positivity for the CRH marker. A CRH Ctvalue <45 was considered a positive result, indicative of the presenceof bladder cancer. In addition to the four bladder cancer markers(KRT20, IGF2, CRH, and ANXA10), the GeneXpert® bladder cancer assayincluded two controls: primers and probe for detecting ABL mRNA in thesamples, and primers and probe for detecting an Armored RNA® exogenouscontrol RNA.

In the first analysis, 132 samples collected from patients who hadpositive cystoscopy results for bladder cancer were tested with theGeneXpert® bladder cancer assay. Sixty of those samples had also beentested using UroVysion®. Table 7 shows the results for those 132samples.

TABLE 7 Assay sensitivity by bladder cancer stage and grade XpertBladder Invalid/ UroVysion POS NEG Error** Sensitivity POS NEGinconclusive Sensitivity Stage: All 94 35 3 72.9% 29 26 5 52.7% Ta,Grade Low 29 23 2 55.8% 5 18 4 21.7% Ta, Grade High 20 2 90.9% 4 6 140.0% T1 13 2 86.7% 6 1 85.7% T2 11 2 84.6% 4 0 100.0% T3 3 0 100.0% 1 0100.0% T4 2 0 100.0% CIS 11 1 91.7% 7 0 100.0% UNK 5 5 1 50.0% 2 1 66.7%Grade: All 94 35 3 72.9% 29 26 5 52.7% Low Grade 33 28 2 54.1% 6 19 424.0% High Grade 61 7 1 89.7% 23 7 1 76.7% **Two invalid results weredue to low ABL Ct, suggesting a low number of cells in the sample, andone of the invalid results was due to poor sample quality.

As shown in Table 7, for these samples, the GeneXpert bladder cancerassay had a sensitivity of 54.1% for low grade bladder cancer and asensitivity of 89.7% for high grade bladder cancer. In contrast,UroVysion® had a sensitivity of just 24% for low grade bladder cancerand a sensitivity of 76.6% for high grade bladder cancer. Further, theGeneXpert® bladder cancer assay was able to detect all grades and stagesof bladder cancer.

The same data set was then divided according to three patient groups:(A) patients with a history of bladder cancer who were currently beingmonitored for recurrence of bladder cancer, (B) patients who had beentreated with Bacillus Calmette-Guerin (BCG) within the three monthsprior to sample collection, and (C) patients who were symptomatic forbladder cancer and had no prior history of bladder cancer. The resultsfor those patient groups are shown in Table 8.

TABLE 8 Assay sensitivity by patient population Xpert Bladder* UroVysionGrade: POS NEG Invalid/Error** Sensitivity POS NEG inconclusiveSensitivity Monitoring (Population A) All 50 20 1 71.4% 11 12 47.8% LowGrade 21 18 53.8% 2 11 1 15.4% High Grade 29 2 1 93.5% 9 1 90.0% Treatedwith BCG in last 3 months (Population B) All 4 2 66.7% 1 100.0% LowGrade High Grade 4 2 66.7% 1 100.0% Symptomatic (Population C) All 40 132 75.5% 17 14 4 54.8% Low Grade 12 10 2 54.5% 4 8 3 33.3% High Grade 283 90.3% 13 6 1 68.4% **Two invalid results were due to low ABL Ct,suggesting a low number of cells in the sample, and one of the invalidresults was due to poor sample quality.

As shown in Table 8, the GeneXpert® bladder cancer assay had a similarsensitivity for low grade and high grade bladder cancer in patientsbeing monitored for bladder cancer and in patients who were symptomaticof bladder cancer as in the patient group as a whole (see Table 7). Inpatients who had been treated with BCG within the last three months, theGeneXpert® bladder cancer assay had a sensitivity of 66.7%, although thesample size was too small (6 samples) to draw any conclusions from thatresult.

In order to have a direct comparison of the GeneXpert® bladder cancerassay and UroVysion®, a dataset was selected that included only samplesthat had been tested with both assays. Table 9 shows the results forthat dataset, with the patients separated into two groups: (A&B)patients with a history of bladder cancer who were currently beingmonitored for recurrence of bladder cancer, combined with patients whohad been treated with Bacillus Calmette-Guerin (BCG) within the threemonths prior to sample collection (these groups were combined becauseonly one sample from a BCG-treated patient had been tested with bothassays), and (C) patients who were symptomatic for bladder cancer andhad no prior history of bladder cancer.

TABLE 9 Assay sensitivity for samples tested with both GeneXpert ® andUroVysion ® Xpert Bladder UroVysion Grade: POS NEG Invalid/Error**Sensitivity POS NEG inconclusive Sensitivity Monitoring and BCG treated(Populations A&B) All 18 7 1 72.0% 12 13 1 48.0% Low Grade 6 7 1 46.2% 212 14.3% High Grade 12 0 100.0% 10 1 1 90.9% Symptomatic (Population C)All 26 8 1 76.5% 17 14 4 54.8% Low Grade 7 7 1 50.0% 4 8 3 33.3% HighGrade 19 1 95.0% 13 6 1 68.4%

As shown in FIG. 9, for those samples that have been tested with bothGeneXpert® bladder cancer assay and UroVysion®, the GeneXpert® bladdercancer assay showed greater sensitivity than UroVysion® for detectinglow grade and high grade cancer in both patient groups.

Next, the data set was divided into samples that had been archived,meaning they were tested more than one week after collection (thesamples ranged from 8 days old up to nine months old), and samples thatwere fresh, meaning they were tested within one week of collection. Theresults of that analysis are shown in Table 10.

TABLE 10 Assay sensitivity in archived and fresh samples Xpert BladderGrade: n POS NEG Invalid/Error** Sensitivity Archived Samples All 89 6125 3 70.9% Low Grade 46 23 21 2 52.3% High Grade 43 38 4 1 90.5% FreshSamples All 43 33 10 76.7% Low Grade 17 10 7 58.8% High Grade 26 23 388.5% **Two invalid results were due to low ABL Ct, suggesting a lownumber of cells in the sample, and one of the invalid results was due topoor sample quality.

As shown in Table 10, the GeneXpert® bladder cancer assay had a similarsensitivity for detecting low grade and high grade bladder cancer infresh and archived samples.

7.3. Example 3: Assay Specificity for Detecting Bladder Cancer UsingMarker Panel KRT20, IGF2, CRH, and ANXA10 in a Larger Cohort

In addition to the samples from patients with positive cystoscopyresults for bladder cancer, urine samples were collected at the sevensites from patients with negative cystoscopy results for bladder cancer,but who were being monitored for recurrence of bladder cancer, hadreceived BCG within the three months prior to sample collection, and whoappeared to be symptomatic for bladder cancer but had no prior historyof bladder cancer. In addition, urine samples were collected frompatients with urology referrals for other suspected conditions, such askidney stones. Finally, urine samples were collected from healthyindividuals. Urine samples were preserved and analyzed using theGeneXpert® bladder cancer assay as described in Example 2.

For the samples from patients with negative cystoscopy results forbladder cancer, the assay results were divided according to the threepatient groups: (A) patients with a history of bladder cancer who werecurrently being monitored for recurrence of bladder cancer, (B) patientswho had been treated with Bacillus Calmette-Guerin (BCG) within thethree months prior to sample collection, and (C) patients who weresymptomatic for bladder cancer and had no prior history of bladdercancer. The results for those patient groups are shown in Table 11.

TABLE 11 Assay specificity in cystoscopy negative patients by populationXpert Bladder POS NEG Invalid/Error Specificity Monitoring (PopulationA) 55 156 17 73.9% BCG Treated (Population B) 1 6 0 85.7% Symptomatic(Population C) 16 86 10 84.3%

As shown in Table 11, the GeneXpert® bladder cancer assay had aspecificity of 73.9%, 85.7%, and 84.3% for patient groups (A), (B), and(C), respectively.

Next, the specificity of the GeneXpert® bladder cancer assay in patientswho were suspected of having other urological conditions, but notbladder cancer, was determined. Seventy patient samples were collectedin this category. The results with the GeneXpert® bladder cancer assaywere 14 positives, 50 negatives, and 6 invalid results. The specificityof the GeneXpert® bladder cancer assay for this patient population wastherefore 78.1%.

Finally, the specificity of the GeneXpert® bladder cancer assay inhealthy individuals was determined. Fifty-five samples were collected inthis category. The results were 4 positives and 51 negatives, indicatinga specificity of 92.7% for this subject category.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that changes can be made without departing fromthe spirit and scope of the invention(s).

TABLE OF CERTAIN SEQUENCES SEQ ID NO Description Sequence 1 Human CRHTCGTTCCTTG GCAGGGCCCT ATGATTTATG CAGGAGCAGA GGCAGCACGC mRNAAATCGAGCTG TCAAGAGAGC GTCAGCTTAT TAGGCAAATG CTGCGTGGTTTTTGAAGAGG GTCGACACTA TAAAATCCCA CTCCAGGCTC TGGAGTGGAGAAACTCAGAG ACCAAGTCCA TTGAGAGACT GAGGGGAAAG AGAGGAGAGAAAGAAAAAGA GAGTGGGAAC AGTAAAGAGA AAGGAAGACA ACCTCCAGAGAAAGCCCCCG GAGACGTCTC TCTGCAGAGA GGCGGCAGCA CCCGGCTCACCTGCGAAGCG CCTGGGAAGC GAGTGCCCCT AACATGCGGC TGCCGCTGCTTGTGTCCGCG GGAGTCCTGC TGGTGGCTCT CCTGCCCTGC CCGCCATGCAGGGCGCTCCT GAGCCGCGGG CCGGTCCCGG GAGCTCGGCA GGCGCCGCAGCACCCTCAGC CCTTGGATTT CTTCCAGCCG CCGCCGCAGT CCGAGCAGCCCCAGCAGCCG CAGGCTCGGC CGGTCCTGCT CCGCATGGGA GAGGAGTACTTCCTCCGCCT GGGGAACCTC AACAAGAGCC CGGCCGCTCC CCTTTCGCCCGCCTCCTCGC TCCTCGCCGG AGGCAGCGGC AGCCGCCCTT CGCCGGAACAGGCGACCGCC AACTTTTTCC GCGTGTTGCT GCAGCAGCTG CTGCTGCCTCGGCGCTCGCT CGACAGCCCC GCGGCTCTCG CGGAGCGCGG CGCTAGGAATGCCCTCGGCG GCCACCAGGA GGCACCGGAG AGAGAAAGGC GGTCCGAGGAGCCTCCCATC TCCCTGGATC TCACCTTCCA CCTCCTCCGG GAAGTCTTGGAAATGGCCAG GGCCGAGCAG TTAGCACAGC AAGCTCACAG CAACAGGAAACTCATGGAGA TTATTGGGAA ATAAAACGGT GCGTTTGGCC AAAAAGAATCTGCATTTAGC ACAAAAAAAA TTTAAAAAAA TACAGTATTC TGTACCATAGCGCTGCTCTT ATGCCATTTG TTTATTTTTA TATAGCTTGA AACATAGAGGGAGAGAGGGA GAGAGCCTAT ACCCCTTACT TAGCATGCAC AAAGTGTATTCACGTGCAGC AGCAACACAA TGTTATTCGT TTTGTCTACG TTTAGTTTCCGTTTCCAGGT GTTTATAGTG GTGTTTTAAA GAGAATGTAG ACCTGTGAGAAAACGTTTTG TTTGAAAAAG CAGACAGAAG TCACTCAATT GTTTTTGTTGTGGTCTGAGC CAAAGAGAAT GCCATTCTCT TGGGTGGGTA AGACTAAATCTGTAAGCTCT TTGAAACAAC TTTCTCTTGT AAACGTTTCA GTAATAAAACATCTTTCCAG TCCTTGGTCA GTTTGGTTGT GTAAGAGAAT GTTGAATACTTATATTTTTA ATAAAAGTTG CAAAGGTAAT CATG 2 Human IGF2CCGCTAATGT ACCATGCCCT GGTGCTGGAA AGTGCCTGAG CCAGCTGCCC mRNA, transcriptCAGCGGCCTC AGCACTACCA AGTTGGCACA AAGCTCCCCA AATTCGGAGG variant 2GGCTCAGGGA AACGAGTGGA GGGGATGAGG AGGTGAGGGG TAAACCCATCATTTCAGTTG GCATTTGAGC AGGTGCCATG CTCAGCGGAG ATGAGGCTCTCCCATCTGTA GGGGCCGTAT TAACATGCAC ACTCTAAAAG TGCCCTTCGTTTCTCCAGCC TCAGCTTTGT CCCTCTCCTC CTCCACGTCA ACCTGGCCAGAGGGTCTGGA CGCCACAGCC AGGGCACCCC CTGCTTTGGT GGTGACTGCTAATATTGGCC AGGCCGGCGG ATCATCGTCC AGGCAGTTTC GGCAGAGAGCCTTGGGCACC AGTGACTCCC CGGTCCTCTT TATCCACTGT CCAGGAGCTGCGGGGACTGC GCAGGGACTA GAGTACAGGG GCCGAAGAGT CACCACCGAGCTTGTGTGGG AGGAGGTGGA TTCCAGCCCC CAGCCCCAGG GCTCTGAATCGCTGCCAGCT CAGCCCCCTG CCCAGCCTGC CCCACAGCCT GAGCCCCAGCAGGCCAGAGA GCCCAGTCCT GAGGTGAGCT GCTGTGGCCT GTGGCCCAGGCGACCCCAGC GCTCCCAGAA CTGAGGCTGG CAGCCAGCCC CAGCCTCAGCCCCAACTGCG AGGCAGAGAG ACACCAATGG GAATCCCAAT GGGGAAGTCGATGCTGGTGC TTCTCACCTT CTTGGCCTTC GCCTCGTGCT GCATTGCTGCTTACCGCCCC AGTGAGACCC TGTGCGGCGG GGAGCTGGTG GACACCCTCCAGTTCGTCTG TGGGGACCGC GGCTTCTACT TCAGCAGGCC CGCAAGCCGTGTGAGCCGTC GCAGCCGTGG CATCGTTGAG GAGTGCTGTT TCCGCAGCTGTGACCTGGCC CTCCTGGAGA CGTACTGTGC TACCCCCGCC AAGTCCGAGAGGGACGTGTC GACCCCTCCG ACCGTGCTTC CGGACAACTT CCCCAGATACCCCGTGGGCA AGTTCTTCCA ATATGACACC TGGAAGCAGT CCACCCAGCGCCTGCGCAGG GGCCTGCCTG CCCTCCTGCG TGCCCGCCGG GGTCACGTGCTCGCCAAGGA GCTCGAGGCG TTCAGGGAGG CCAAACGTCA CCGTCCCCTGATTGCTCTAC CCACCCAAGA CCCCGCCCAC GGGGGCGCCC CCCCAGAGATGGCCAGCAAT CGGAAGTGAG CAAAACTGCC GCAAGTCTGC AGCCCGGCGCCACCATCCTG CAGCCTCCTC CTGACCACGG ACGTTTCCAT CAGGTTCCATCCCGAAAATC TCTCGGTTCC ACGTCCCCCT GGGGCTTCTC CTGACCCAGTCCCCGTGCCC CGCCTCCCCG AAACAGGCTA CTCTCCTCGG CCCCCTCCATCGGGCTGAGG AAGCACAGCA GCATCTTCAA ACATGTACAA AATCGATTGGCTTTAAACAC CCTTCACATA CCCTCCCCCC AAATTATCCC CAATTATCCCCACACATAAA AAATCAAAAC ATTAAACTAA CCCCCTTCCC CCCCCCCCACAACAACCCTC TTAAAACTAA TTGGCTTTTT AGAAACACCC CACAAAAGCTCAGAAATTGG CTTTAAAAAA AACAACCACC AAAAAAAATC AATTGGCTAAAAAAAAAAAG TATTAAAAAC GAATTGGCTG AGAAACAATT GGCAAAATAAAGGAATTTGG CACTCCCCAC CCCCCTCTTT CTCTTCTCCC TTGGACTTTGAGTCAAATTG GCCTGGACTT GAGTCCCTGA ACCAGCAAAG AGAAAAGAAGGACCCCAGAA ATCACAGGTG GGCACGTCGC TGCTACCGCC ATCTCCCTTCTCACGGGAAT TTTCAGGGTA AACTGGCCAT CCGAAAATAG CAACAACCCAGACTGGCTCC TCACTCCCTT TTCCATCACT AAAAATCACA GAGCAGTCAGAGGGACCCAG TAAGACCAAA GGAGGGGAGG ACAGAGCATG AAAACCAAAATCCATGCAAA TGAAATGTAA TTGGCACGAC CCTCACCCCC AAATCTTACATCTCAATTCC CATCCTAAAA AGCACTCATA CTTTATGCAT CCCCGCAGCTACACACACAC AACACACAGC ACACGCATGA ACACAGCACA CACACGAGCACAGCACACAC ACAAACGCAC AGCACACACA GCACACAGAT GAGCACACAGCACACACACA AACGCACAGC ACACACACGC ACACACATGC ACACACAGCACACAAACGCA CGGCACACAC ACGCACACAC ATGCACACAC AGCACACACACAAACGCACA GCACACACAA ACGCACAGCA CACACGCACA CACAGCACACACACGAGCAC ACAGCACACA AACGCACAGC ACACGCACAC ACATGCACACACAGCACACA CACTAGCACA CAGCACACAC ACAAAGACAC AGCACACACATGCACACACA GCACACACAC GCGAACACAG CACACACGAA CACAGCACACACAGCACACA CACAAACACA GCACACACAT GCACACAGCA CACGCACACACACGACACAC ATGAACACAG CACACAGCAC ACACATGCAC ACACAGCACACACGCATGCA CAGCACACAT GAACACAGCA CACACACAAA CACACAGCACACACATGCAC ACACAGCACA CACACTCATG CGCAGCACAT ACATGAACACAGCTCACAGC ACACAAACAC GCAGCACACA CGTTGCACAC GCAAGCACCCACCTGCACAC ACACATGCGC ACACACACGC ACACCCCCAC AAAATTGGATGAAAACAATA AGCATATCTA AGCAACTACG ATATCTGTAT GGATCAGGCCAAAGTCCCGC TAAGATTCTC CAATGTTTTC ATGGTCTGAG CCCCGCTCCTGTTCCCATCT CCACTGCCCC TCGGCCCTGT CTGTGCCCTG CCTCTCAGAGGAGGGGGCTC AGATGGTGCG GCCTGAGTGT GCGGCCGGCG GCATTTGGGATACACCCGTA GGGTGGGCGG GGTGTGTCCC AGGCCTAATT CCATCTTTCCACCATGACAG AGATGCCCTT GTGAGGCTGG CCTCCTTGGC GCCTGTCCCCACGGCCCCCG CAGCGTGAGC CACGATGCTC CCCATACCCC ACCCATTCCCGATACACCTT ACTTACTGTG TGTTGGCCCA GCCAGAGTGA GGAAGGAGTTTGGCCACATT GGAGATGGCG GTAGCTGAGC AGACATGCCC CCACGAGTAGCCTGACTCCC TGGTGTGCTC CTGGAAGGAA GATCTTGGGG ACCCCCCCACCGGAGCACAC CTAGGGATCA TCTTTGCCCG TCTCCTGGGG ACCCCCCAAGAAATGTGGAG TCCTCGGGGG CCGTGCACTG ATGCGGGGAG TGTGGGAAGTCTGGCGGTTG GAGGGGTGGG TGGGGGGCAG TGGGGGCTGG GCGGGGGGAGTTCTGGGGTA GGAAGTGGTC CCGGGAGATT TTGGATGGAA AAGTCAGGAGGATTGACAGC AGACTTGCAG AATTACATAG AGAAATTAGG AACCCCCAAATTTCATGTCA ATTGATCTAT TCCCCCTCTT TGTTTCTTGG GGCATTTTTCCTTTTTTTTT TTTTTTTGTT TTTTTTTTAC CCCTCCTTAG CTTTATGCGCTCAGAAACCA AATTAAACCC CCCCCCCATG TAACAGGGGG GCAGTGACAAAAGCAAGAAC GCACGAAGCC AGCCTGGAGA CCACCACGTC CTGCCCCCCGCCATTTATCG CCCTGATTGG ATTTTGTTTT TCATCTGTCC CTGTTGCTTGGGTTGAGTTG AGGGTGGAGC CTCCTGGGGG GCACTGGCCA CTGAGCCCCCTTGGAGAAGT CAGAGGGGAG TGGAGAAGGC CACTGTCCGG CCTGGCTTCTGGGGACAGTG GCTGGTCCCC AGAAGTCCTG AGGGCGGAGG GGGGGGTTGGGCAGGGTCTC CTCAGGTGTC AGGAGGGTGC TCGGAGGCCA CAGGAGGGGGCTCCTGGCTG GCCTGAGGCT GGCCGGAGGG GAAGGGGCTA GCAGGTGTGTAAACAGAGGG TTCCATCAGG CTGGGGCAGG GTGGCCGCCT TCCGCACACTTGAGGAACCC TCCCCTCTCC CTCGGTGACA TCTTGCCCGC CCCTCAGCACCCTGCCTTGT CTCCAGGAGG TCCGAAGCTC TGTGGGACCT CTTGGGGGCAAGGTGGGGTG AGGCCGGGGA GTAGGGAGGT CAGGCGGGTC TGAGCCCACAGAGCAGGAGA GCTGCCAGGT CTGCCCATCG ACCAGGTTGC TTGGGCCCCGGAGCCCACGG GTCTGGTGAT GCCATAGCAG CCACCACCGC GGCGCCTAGGGCTGCGGCAG GGACTCGGCC TCTGGGAGGT TTACCTCGCC CCCACTTGTGCCCCCAGCTC AGCCCCCCTG CACGCAGCCC GACTAGCAGT CTAGAGGCCTGAGGCTTCTG GGTCCTGGTG ACGGGGCTGG CATGACCCCG GGGGTCGTCCATGCCAGTCC GCCTCAGTCG CAGAGGGTCC CTCGGCAAGC GCCCTGTGAGTGGGCCATTC GGAACATTGG ACAGAAGCCC AAAGAGCCAA ATTGTCACAATTGTGGAACC CACATTGGCC TGAGATCCAA AACGCTTCGA GGCACCCCAAATTACCTGCC CATTCGTCAG GACACCCACC CACCCAGTGT TATATTCTGCCTCGCCGGAG TGGGTGTTCC CGGGGGCACT TGCCGACCAG CCCCTTGCGTCCCCAGGTTT GCAGCTCTCC CCTGGGCCAC TAACCATCCT GGCCCGGGCTGCCTGTCTGA CCTCCGTGCC TAGTCGTGGC TCTCCATCTT GTCTCCTCCCCGTGTCCCCA ATGTCTTCAG TGGGGGGCCC CCTCTTGGGT CCCCTCCTCTGCCATCACCT GAAGACCCCC ACGCCAAACA CTGAATGTCA CCTGTGCCTGCCGCCTCGGT CCACCTTGCG GCCCGTGTTT GACTCAACTC AACTCCTTTAACGCTAATAT TTCCGGCAAA ATCCCATGCT TGGGTTTTGT CTTTAACCTTGTAACGCTTG CAATCCCAAT AAAGCATTAA AAGTCATGAA AAAAAAAAAA AAAAAA 3Human IGF2 CGCCTGTCCC CCTCCCGAGG CCCGGGCTCG CGACGGCAGA GGGCTCCGTCmRNA, transcript GGCCCAAACC GAGCTGGGCG CCCGCGGTCC GGGTGCAGCC TCCACTCCGCvariant 2 CCCCCAGTCA CCGCCTCCCC CGGCCCCTCG ACGTGGCGCC CTTCCCTCCGCTTCTCTGTG CTCCCCGCGC CCCTCTTGGC GTCTGGCCCC GGCCCCCGCTCTTTCTCCCG CAACCTTCCC TTCGCTCCCT CCCGTCCCCC CCAGCTCCTAGCCTCCGACT CCCTCCCCCC CTCACGCCCG CCCTCTCGCC TTCGCCGAACCAAAGTGGAT TAATTACACG CTTTCTGTTT CTCTCCGTGC TGTTCTCTCCCGCTGTGCGC CTGCCCGCCT CTCGCTGTCC TCTCTCCCCC TCGCCCTCTCTTCGGCCCCC CCCTTTCACG TTCACTCTGT CTCTCCCACT ATCTCTGCCCCCCTCTATCC TTGATACAAC AGCTGACCTC ATTTCCCGAT ACCTTTTCCCCCCCGAAAAG TACAACATCT GGCCCGCCCC AGCCCGAAGA CAGCCCGTCCTCCCTGGACA ATCAGACGAA TTCTCCCCCC CCCCCCAAAA AAAAGCCATCTCCCTGGACA ATCAGACGAA TTCTCCCCCC CCCCCCAAAA AAAAGCCATCCCCCCGCTCT GCCCCGTCGC ACATTCGGCC CCCGCGACTC GGCCAGAGCGGCGCTGGCAG AGGAGTGTCC GGCAGGAGGG CCAACGCCCG CTGTTCGGTTTGCGACACGC AGCAGGGAGG TGGGCGGCAG CGTCGCCGGC TTCCAGACACCAATGGGAAT CCCAATGGGG AAGTCGATGC TGGTGCTTCT CACCTTCTTGGCCTTCGCCT CGTGCTGCAT TGCTGCTTAC CGCCCCAGTG AGACCCTGTGCGGCGGGGAG CTGGTGGACA CCCTCCAGTT CGTCTGTGGG GACCGCGGCTTCTACTTCAG CAGGCCCGCA AGCCGTGTGA GCCGTCGCAG CCGTGGCATCGTTGAGGAGT GCTGTTTCCG CAGCTGTGAC CTGGCCCTCC TGGAGACGTACTGTGCTACC CCCGCCAAGT CCGAGAGGGA CGTGTCGACC CCTCCGACCGTGCTTCCGGA CAACTTCCCC AGATACCCCG TGGGCAAGTT CTTCCAATATGACACCTGGA AGCAGTCCAC CCAGCGCCTG CGCAGGGGCC TGCCTGCCCTCCTGCGTGCC CGCCGGGGTC ACGTGCTCGC CAAGGAGCTC GAGGCGTTCAGGGAGGCCAA ACGTCACCGT CCCCTGATTG CTCTACCCAC CCAAGACCCCGCCCACGGGG GCGCCCCCCC AGAGATGGCC AGCAATCGGA AGTGAGCAAAACTGCCGCAA GTCTGCAGCC CGGCGCCACC ATCCTGCAGC CTCCTCCTGACCACGGACGT TTCCATCAGG TTCCATCCCG AAAATCTCTC GGTTCCACGTCCCCCTGGGG CTTCTCCTGA CCCAGTCCCC GTGCCCCGCC TCCCCGAAACAGGCTACTCT CCTCGGCCCC CTCCATCGGG CTGAGGAAGC ACAGCAGCATCTTCAAACAT GTACAAAATC GATTGGCTTT AAACACCCTT CACATACCCTCCCCCCAAAT TATCCCCAAT TATCCCCACA CATAAAAAAT CAAAACATTAAACTAACCCC CTTCCCCCCC CCCCACAACA ACCCTCTTAA AACTAATTGGCTTTTTAGAA ACACCCCACA AAAGCTCAGA AATTGGCTTT AAAAAAAACAACCACCAAAA AAAATCAATT GGCTAAAAAA AAAAAGTATT AAAAACGAATTGGCTGAGAA ACAATTGGCA AAATAAAGGA ATTTGGCACT CCCCACCCCCCTCTTTCTCT TCTCCCTTGG ACTTTGAGTC AAATTGGCCT GGACTTGAGTCCCTGAACCA GCAAAGAGAA AAGAAGGACC CCAGAAATCA CAGGTGGGCACGTCGCTGCT ACCGCCATCT CCCTTCTCAC GGGAATTTTC AGGGTAAACTGGCCATCCGA AAATAGCAAC AACCCAGACT GGCTCCTCAC TCCCTTTTCCATCACTAAAA ATCACAGAGC AGTCAGAGGG ACCCAGTAAG ACCAAAGGAGGGGAGGACAG AGCATGAAAA CCAAAATCCA TGCAAATGAA ATGTAATTGGCACGACCCTC ACCCCCAAAT CTTACATCTC AATTCCCATC CTAAAAAGCACTCATACTTT ATGCATCCCC GCAGCTACAC ACACACAACA CACAGCACACGCATGAACAC AGCACACACA CGAGCACAGC ACACACACAA ACGCACAGCACACACAGCAC ACAGATGAGC ACACAGCACA CACACAAACG CACAGCACACACACGCACAC ACATGCACAC ACAGCACACA AACGCACGGC ACACACACGCACACACATGC ACACACAGCA CACACACAAA CGCACAGCAC ACACAAACGCACAGCACACA CGCACACACA GCACACACAC GAGCACACAG CACACAAACGCACAGCACAC GCACACACAT GCACACACAG CACACACACT AGCACACAGCACACACACAA AGACACAGCA CACACATGCA CACACAGCAC ACACACGCGAACACAGCACA CACGAACACA GCACACACAG CACACACACA AACACAGCACACACATGCAC ACAGCACACG CACACACAGC ACACACATGA ACACAGCACACAGCACACAC ATGCACACAC AGCACACACG CATGCACAGC ACACATGAACACAGCACACA CACAAACACA CAGCACACAC ATGCACACAC AGCACACACACTCATGCGCA GCACATACAT GAACACAGCT CACAGCACAC AAACACGCAGCACACACGTT GCACACGCAA GCACCCACCT GCACACACAC ATGCGCACACACACGCACAC CCCCACAAAA TTGGATGAAA ACAATAAGCA TATCTAAGCAACTACGATAT CTGTATGGAT CAGGCCAAAG TCCCGCTAAG ATTCTCCAATGTTTTCATGG TCTGAGCCCC GCTCCTGTTC CCATCTCCAC TGCCCCTCGGCCCTGTCTGT GCCCTGCCTC TCAGAGGAGG GGGCTCAGAT GGTGCGGCCTGAGTGTGCGG CCGGCGGCAT TTGGGATACA CCCGTAGGGT GGGCGGGGTGTGTCCCAGGC CTAATTCCAT CTTTCCACCA TGACAGAGAT GCCCTTGTGAGGCTGGCCTC CTTGGCGCCT GTCCCCACGG CCCCCGCAGC GTGAGCCACGATGCTCCCCA TACCCCACCC ATTCCCGATA CACCTTACTT ACTGTGTGTTGGCCCAGCCA GAGTGAGGAA GGAGTTTGGC CACATTGGAG ATGGCGGTAGCTGAGCAGAC ATGCCCCCAC GAGTAGCCTG ACTCCCTGGT GTGCTCCTGGAAGGAAGATC TTGGGGACCC CCCCACCGGA GCACACCTAG GGATCATCTTTGCCCGTCTC CTGGGGACCC CCCAAGAAAT GTGGAGTCCT CGGGGGCCGTGCACTGATGC GGGGAGTGTG GGAAGTCTGG CGGTTGGAGG GGTGGGTGGGGGGCAGTGGG GGCTGGGCGG GGGGAGTTCT GGGGTAGGAA GTGGTCCCGGGAGATTTTGG ATGGAAAAGT CAGGAGGATT GACAGCAGAC TTGCAGAATTACATAGAGAA ATTAGGAACC CCCAAATTTC ATGTCAATTG ATCTATTCCCCCTCTTTGTT TCTTGGGGCA TTTTTCCTTT TTTTTTTTTT TTTGTTTTTTTTTTACCCCT CCTTAGCTTT ATGCGCTCAG AAACCAAATT AAACCCCCCCCCCATGTAAC AGGGGGGCAG TGACAAAAGC AAGAACGCAC GAAGCCAGCCTGGAGACCAC CACGTCCTGC CCCCCGCCAT TTATCGCCCT GATTGGATTTTGTTTTTCAT CTGTCCCTGT TGCTTGGGTT GAGTTGAGGG TGGAGCCTCCTGGGGGGCAC TGGCCACTGA GCCCCCTTGG AGAAGTCAGA GGGGAGTGGAGAAGGCCACT GTCCGGCCTG GCTTCTGGGG ACAGTGGCTG GTCCCCAGAAGTCCTGAGGG CGGAGGGGGG GGTTGGGCAG GGTCTCCTCA GGTGTCAGGAGGGTGCTCGG AGGCCACAGG AGGGGGCTCC TGGCTGGCCT GAGGCTGGCCGGAGGGGAAG GGGCTAGCAG GTGTGTAAAC AGAGGGTTCC ATCAGGCTGGGGCAGGGTGG CCGCCTTCCG CACACTTGAG GAACCCTCCC CTCTCCCTCGGTGACATCTT GCCCGCCCCT CAGCACCCTG CCTTGTCTCC AGGAGGTCCGAAGCTCTGTG GGACCTCTTG GGGGCAAGGT GGGGTGAGGC CGGGGAGTAGGGAGGTCAGG CGGGTCTGAG CCCACAGAGC AGGAGAGCTG CCAGGTCTGCCCATCGACCA GGTTGCTTGG GCCCCGGAGC CCACGGGTCT GGTGATGCCATAGCAGCCAC CACCGCGGCG CCTAGGGCTG CGGCAGGGAC TCGGCCTCTGGGAGGTTTAC CTCGCCCCCA CTTGTGCCCC CAGCTCAGCC CCCCTGCACGCAGCCCGACT AGCAGTCTAG AGGCCTGAGG CTTCTGGGTC CTGGTGACGGGGCTGGCATG ACCCCGGGGG TCGTCCATGC CAGTCCGCCT CAGTCGCAGAGGGTCCCTCG GCAAGCGCCC TGTGAGTGGG CCATTCGGAA CATTGGACAGAAGCCCAAAG AGCCAAATTG TCACAATTGT GGAACCCACA TTGGCCTGAGATCCAAAACG CTTCGAGGCA CCCCAAATTA CCTGCCCATT CGTCAGGACACCCACCCACC CAGTGTTATA TTCTGCCTCG CCGGAGTGGG TGTTCCCGGGGGCACTTGCC GACCAGCCCC TTGCGTCCCC AGGTTTGCAG CTCTCCCCTGGGCCACTAAC CATCCTGGCC CGGGCTGCCT GTCTGACCTC CGTGCCTAGTCGTGGCTCTC CATCTTGTCT CCTCCCCGTG TCCCCAATGT CTTCAGTGGGGGGCCCCCTC TTGGGTCCCC TCCTCTGCCA TCACCTGAAG ACCCCCACGCCAAACACTGA ATGTCACCTG TGCCTGCCGC CTCGGTCCAC CTTGCGGCCCGTGTTTGACT CAACTCAACT CCTTTAACGC TAATATTTCC GGCAAAATCCCATGCTTGGG TTTTGTCTTT AACCTTGTAA CGCTTGCAAT CCCAATAAAGCATTAAAAGT CATGAAAAAA AAAAAAAAAA AA 4 Human IGF2GGCCGCGCGC CCTCAGGACG TGGACAGGGA GGGCTTCCCC GTGTCCAGGA mRNA, transcriptAAGCGACCGG GCATTGCCCC CAGTCTCCCC CAAATTTGGG CATTGTCCCC variant 3GGGTCTTCCA ACGGACTGGG CGTTGCTCCC GGACACTGAG GACTGGCCCCGGGGTCTCGC TCACCTTCAG CAGCGTCCAC CGCCTGCCAC AGAGCGTTCGATCGCTCGCT GCCTGAGCTC CTGGTGCGCC CGCGGACGCA GCCTCCAGCTTCGCGGAGAT GGTTTCCCCA GACCCCCAAA TTATCGTGGT GGCCCCCGAGACCGAACTCG CGTCTATGCA AGTCCAACGC ACTGAGGACG GGGTAACCATTATCCAGATA TTTTGGGTGG GCCGCAAAGG CGAGCTACTT AGACGCACCCCGGTGAGCTC GGCCATGCAG ACACCAATGG GAATCCCAAT GGGGAAGTCGATGCTGGTGC TTCTCACCTT CTTGGCCTTC GCCTCGTGCT GCATTGCTGCTTACCGCCCC AGTGAGACCC TGTGCGGCGG GGAGCTGGTG GACACCCTCCAGTTCGTCTG TGGGGACCGC GGCTTCTACT TCAGCAGGCC CGCAAGCCGTGTGAGCCGTC GCAGCCGTGG CATCGTTGAG GAGTGCTGTT TCCGCAGCTGTGACCTGGCC CTCCTGGAGA CGTACTGTGC TACCCCCGCC AAGTCCGAGAGGGACGTGTC GACCCCTCCG ACCGTGCTTC CGGACAACTT CCCCAGATACCCCGTGGGCA AGTTCTTCCA ATATGACACC TGGAAGCAGT CCACCCAGCGCCTGCGCAGG GGCCTGCCTG CCCTCCTGCG TGCCCGCCGG GGTCACGTGCTCGCCAAGGA GCTCGAGGCG TTCAGGGAGG CCAAACGTCA CCGTCCCCTGATTGCTCTAC CCACCCAAGA CCCCGCCCAC GGGGGCGCCC CCCCAGAGATGGCCAGCAAT CGGAAGTGAG CAAAACTGCC GCAAGTCTGC AGCCCGGCGCCACCATCCTG CAGCCTCCTC CTGACCACGG ACGTTTCCAT CAGGTTCCATCCCGAAAATC TCTCGGTTCC ACGTCCCCCT GGGGCTTCTC CTGACCCAGTCCCCGTGCCC CGCCTCCCCG AAACAGGCTA CTCTCCTCGG CCCCCTCCATCGGGCTGAGG AAGCACAGCA GCATCTTCAA ACATGTACAA AATCGATTGGCTTTAAACAC CCTTCACATA CCCTCCCCCC AAATTATCCC CAATTATCCCCACACATAAA AAATCAAAAC ATTAAACTAA CCCCCTTCCC CCCCCCCCACAACAACCCTC TTAAAACTAA TTGGCTTTTT AGAAACACCC CACAAAAGCTCAGAAATTGG CTTTAAAAAA AACAACCACC AAAAAAAATC AATTGGCTAAAAAAAAAAAG TATTAAAAAC GAATTGGCTG AGAAACAATT GGCAAAATAAAGGAATTTGG CACTCCCCAC CCCCCTCTTT CTCTTCTCCC TTGGACTTTGAGTCAAATTG GCCTGGACTT GAGTCCCTGA ACCAGCAAAG AGAAAAGAAGGACCCCAGAA ATCACAGGTG GGCACGTCGC TGCTACCGCC ATCTCCCTTCTCACGGGAAT TTTCAGGGTA AACTGGCCAT CCGAAAATAG CAACAACCCAGACTGGCTCC TCACTCCCTT TTCCATCACT AAAAATCACA GAGCAGTCAGAGGGACCCAG TAAGACCAAA GGAGGGGAGG ACAGAGCATG AAAACCAAAATCCATGCAAA TGAAATGTAA TTGGCACGAC CCTCACCCCC AAATCTTACATCTCAATTCC CATCCTAAAA AGCACTCATA CTTTATGCAT CCCCGCAGCTACACACACAC AACACACAGC ACACGCATGA ACACAGCACA CACACGAGCACAGCACACAC ACAAACGCAC AGCACACACA GCACACAGAT GAGCACACAGCACACACACA AACGCACAGC ACACACACGC ACACACATGC ACACACAGCACACAAACGCA CGGCACACAC ACGCACACAC ATGCACACAC AGCACACACACAAACGCACA GCACACACAA ACGCACAGCA CACACGCACA CACAGCACACACACGAGCAC ACAGCACACA AACGCACAGC ACACGCACAC ACATGCACACACAGCACACA CACTAGCACA CAGCACACAC ACAAAGACAC AGCACACACATGCACACACA GCACACACAC GCGAACACAG CACACACGAA CACAGCACACACAGCACACA CACAAACACA GCACACACAT GCACACAGCA CACGCACACACAGCACACAC ATGAACACAG CACACAGCAC ACACATGCAC ACACAGCACACACGCATGCA CAGCACACAT GAACACAGCA CACACACAAA CACACAGCACACACATGCAC ACACAGCACA CACACTCATG CGCAGCACAT ACATGAACACAGCTCACAGC ACACAAACAC GCAGCACACA CGTTGCACAC GCAAGCACCCACCTGCACAC ACACATGCGC ACACACACGC ACACCCCCAC AAAATTGGATGAAAACAATA AGCATATCTA AGCAACTACG ATATCTGTAT GGATCAGGCCAAAGTCCCGC TAAGATTCTC CAATGTTTTC ATGGTCTGAG CCCCGCTCCTGTTCCCATCT CCACTGCCCC TCGGCCCTGT CTGTGCCCTG CCTCTCAGAGGAGGGGGCTC AGATGGTGCG GCCTGAGTGT GCGGCCGGCG GCATTTGGGATACACCCGTA GGGTGGGCGG GGTGTGTCCC AGGCCTAATT CCATCTTTCCACCATGACAG AGATGCCCTT GTGAGGCTGG CCTCCTTGGC GCCTGTCCCCACGGCCCCCG CAGCGTGAGC CACGATGCTC CCCATACCCC ACCCATTCCCGATACACCTT ACTTACTGTG TGTTGGCCCA GCCAGAGTGA GGAAGGAGTTTGGCCACATT GGAGATGGCG GTAGCTGAGC AGACATGCCC CCACGAGTAGCCTGACTCCC TGGTGTGCTC CTGGAAGGAA GATCTTGGGG ACCCCCCCACCGGAGCACAC CTAGGGATCA TCTTTGCCCG TCTCCTGGGG ACCCCCCAAGAAATGTGGAG TCCTCGGGGG CCGTGCACTG ATGCGGGGAG TGTGGGAAGTCTGGCGGTTG GAGGGGTGGG TGGGGGGCAG TGGGGGCTGG GCGGGGGGAGTTCTGGGGTA GGAAGTGGTC CCGGGAGATT TTGGATGGAA AAGTCAGGAGGATTGACAGC AGACTTGCAG AATTACATAG AGAAATTAGG AACCCCCAAATTTCATGTCA ATTGATCTAT TCCCCCTCTT TGTTTCTTGG GGCATTTTTCCTTTTTTTTT TTTTTTTGTT TTTTTTTTAC CCCTCCTTAG CTTTATGCGCTCAGAAACCA AATTAAACCC CCCCCCCATG TAACAGGGGG GCAGTGACAAAAGCAAGAAC GCACGAAGCC AGCCTGGAGA CCACCACGTC CTGCCCCCCGCCATTTATCG CCCTGATTGG ATTTTGTTTT TCATCTGTCC CTGTTGCTTGGGTTGAGTTG AGGGTGGAGC CTCCTGGGGG GCACTGGCCA CTGAGCCCCCTTGGAGAAGT CAGAGGGGAG TGGAGAAGGC CACTGTCCGG CCTGGCTTCTGGGGACAGTG GCTGGTCCCC AGAAGTCCTG AGGGCGGAGG GGGGGGTTGGGCAGGGTCTC CTCAGGTGTC AGGAGGGTGC TCGGAGGCCA CAGGAGGGGGCTCCTGGCTG GCCTGAGGCT GGCCGGAGGG GAAGGGGCTA GCAGGTGTGTAAACAGAGGG TTCCATCAGG CTGGGGCAGG GTGGCCGCCT TCCGCACACTTGAGGAACCC TCCCCTCTCC CTCGGTGACA TCTTGCCCGC CCCTCAGCACCCTGCCTTGT CTCCAGGAGG TCCGAAGCTC TGTGGGACCT CTTGGGGGCAAGGTGGGGTG AGGCCGGGGA GTAGGGAGGT CAGGCGGGTC TGAGCCCACAGAGCAGGAGA GCTGCCAGGT CTGCCCATCG ACCAGGTTGC TTGGGCCCCGGAGCCCACGG GTCTGGTGAT GCCATAGCAG CCACCACCGC GGCGCCTAGGGCTGCGGCAG GGACTCGGCC TCTGGGAGGT TTACCTCGCC CCCACTTGTGCCCCCAGCTC AGCCCCCCTG CACGCAGCCC GACTAGCAGT CTAGAGGCCTGAGGCTTCTG GGTCCTGGTG ACGGGGCTGG CATGACCCCG GGGGTCGTCCATGCCAGTCC GCCTCAGTCG CAGAGGGTCC CTCGGCAAGC GCCCTGTGAGTGGGCCATTC GGAACATTGG ACAGAAGCCC AAAGAGCCAA ATTGTCACAATTGTGGAACC CACATTGGCC TGAGATCCAA AACGCTTCGA GGCACCCCAAATTACCTGCC CATTCGTCAG GACACCCACC CACCCAGTGT TATATTCTGCCTCGCCGGAG TGGGTGTTCC CGGGGGCACT TGCCGACCAG CCCCTTGCGTCCCCAGGTTT GCAGCTCTCC CCTGGGCCAC TAACCATCCT GGCCCGGGCTGCCTGTCTGA CCTCCGTGCC TAGTCGTGGC TCTCCATCTT GTCTCCTCCCCGTGTCCCCA ATGTCTTCAG TGGGGGGCCC CCTCTTGGGT CCCCTCCTCTGCCATCACCT GAAGACCCCC ACGCCAAACA CTGAATGTCA CCTGTGCCTGCCGCCTCGGT CCACCTTGCG GCCCGTGTTT GACTCAACTC AACTCCTTTAACGCTAATAT TTCCGGCAAA ATCCCATGCT TGGGTTTTGT CTTTAACCTTGTAACGCTTG CAATCCCAAT AAAGCATTAA AAGTCATGAA AAAAAAAAAA AAAAAA 5Human KRT20 GAGACACACT CTGCCCCAAC CATCCTGAAG CTACAGGTGC TCCCTCCTGG mRNAAATCTCCAAT GGATTTCAGT CGCAGAAGCT TCCACAGAAG CCTGAGCTCCTCCTTGCAGG CCCCTGTAGT CAGTACAGTG GGCATGCAGC GCCTCGGGACGACACCCAGC GTTTATGGGG GTGCTGGAGG CCGGGGCATC CGCATCTCCAACTCCAGACA CACGGTGAAC TATGGGAGCG ATCTCACAGG CGGCGGGGACCTGTTTGTTG GCAATGAGAA AATGGCCATG CAGAACCTAA ATGACCGTCTAGCGAGCTAC CTAGAAAAGG TGCGGACCCT GGAGCAGTCC AACTCCAAACTTGAAGTGCA AATCAAGCAG TGGTACGAAA CCAACGCCCC GAGGGCTGGTCGCGACTACA GTGCATATTA CAGACAAATT GAAGAGCTGC GAAGTCAGATTAAGGATGCT CAACTGCAAA ATGCTCGGTG TGTCCTGCAA ATTGATAATGCTAAACTGGC TGCTGAGGAC TTCAGACTGA AGTATGAGAC TGAGAGAGGAATACGTCTAA CAGTGGAAGC TGATCTCCAA GGCCTGAATA AGGTCTTTGATGACCTAACC CTACATAAAA CAGATTTGGA GATTCAAATT GAAGAACTGAATAAAGACCT AGCTCTCCTC AAAAAGGAGC ATCAGGAGGA AGTCGATGGCCTACACAAGC ATCTGGGCAA CACTGTCAAT GTGGAGGTTG ATGCTGCTCCAGGCCTGAAC CTTGGCGTCA TCATGAATGA AATGAGGCAG AAGTATGAAGTCATGGCCCA GAAGAACCTT CAAGAGGCCA AAGAACAGTT TGAGAGACAGACTGCAGTTC TGCAGCAACA GGTCACAGTG AATACTGAAG AATTAAAAGGAACTGAGGTT CAACTAACGG AGCTGAGACG CACCTCCCAG AGCCTTGAGATAGAACTCCA GTCCCATCTC AGCATGAAAG AGTCTTTGGA GCACACTCTAGAGGAGACCA AGGCCCGTTA CAGCAGCCAG TTAGCCAACC TCCAGTCGCTGTTGAGCTCT CTGGAGGCCC AACTGATGCA GATTCGGAGT AACATGGAACGCCAGAACAA CGAATACCAT ATCCTTCTTG ACATAAAGAC TCGACTTGAACAGGAAATTG CTACTTACCG CCGCCTTCTG GAAGGAGAAG ACGTAAAAACTACAGAATAT CAGTTAAGCA CCCTGGAAGA GAGAGATATA AAGAAAACCAGGAAGATTAA GACAGTCGTG CAAGAAGTAG TGGATGGCAA GGTCGTGTCATCTGAAGTCA AAGAGGTGGA AGAAAATATC TAAATAGCTA CCAGAAGGAGATGCTGCTGA GGTTTTGAAA GAAATTTGGC TATAATCTTA TCTTTGCTCCCTGCAAGAAA TCAGCCATAA GAAAGCACTA TTAATACTCT GCAGTGATTAGAAGGGGTGG GGTGGCGGGA ATCCTATTTA TCAGACTCTG TAATTGAATATAAATGTTTT ACTCAGAGGA GCTGCAAATT GCCTGCAAAA ATGAAATCCAGTGAGCACTA GAATATTTAA AACATCATTA CTGCCATCTT TATCATGAAGCACATCAATT ACAAGCTGTA GACCACCTAA TATCAATTTG TAGGTAATGTTCCTGAAAAT TGCAATACAT TTCAATTATA CTAAACCTCA CAAAGTAGAGGAATCCATGT AAATTGCAAA TAAACCACTT TCTAATTTTT TCCTGTTTCTGAATTGTAAA ACCCCCTTTG GGAGTCCCTG GTTTCTTATT GAGCCAATTT CTGGG 6Human ANXA10 ATCCAGATTT GCTTTTACAT TTTCTTGCCT GAGTCTGAGG TGAACAGTGA mRNAACATATTTAC ATTTGATTTA ACAGTGAACC TTAATTCTTT CTGGCTTCACAGTGAAACAA GTTTATGCAA TCGATCAAAT ATTTTCATCC CTGAGGTTAACAATTACCAT CAAAATGTTT TGTGGAGACT ATGTGCAAGG AACCATCTTCCCAGCTCCCA ATTTCAATCC CATAATGGAT GCCCAAATGC TAGGAGGAGCACTCCAAGGA TTTGACTGTG ACAAAGACAT GCTGATCAAC ATTCTGACTCAGCGCTGCAA TGCACAAAGG ATGATGATTG CAGAGGCATA CCAGAGCATGTATGGCCGGG ACCTGATTGG GGATATGAGG GAGCAGCTTT CGGATCACTTCAAAGATGTG ATGGCTGGCC TCATGTACCC ACCACCACTG TATGATGCTCATGAGCTCTG GCATGCCATG AAGGGAGTAG GCACTGATGA GAATTGCCTCATTGAAATAC TAGCTTCAAG AACAAATGGA GAAATTTTCC AGATGCGAGAAGCCTACTGC TTGCAATACA GCAATAACCT CCAAGAGGAC ATTTATTCAGAGACCTCAGG ACACTTCAGA GATACTCTCA TGAACTTGGT CCAGGGGACCAGAGAGGAAG GATATACAGA CCCTGCGATG GCTGCTCAGG ATGCAATGGTCCTATGGGAA GCCTGTCAGC AGAAGACGGG GGAGCACAAA ACCATGCTGCAAATGATCCT GTGCAACAAG AGCTACCAGC AGCTGCGGCT GGTTTTCCAGGAATTTCAAA ATATTTCTGG GCAAGATATG GTAGATGCCA TTAATGAATGTTATGATGGA TACTTTCAGG AGCTGCTGGT TGCAATTGTT CTCTGTGTTCGAGACAAACC AGCCTATTTT GCTTATAGAT TATATAGTGC AATTCATGACTTTGGTTTCC ATAATAAAAC TGTAATCAGG ATTCTCATTG CCAGAAGTGAAATAGACCTG CTGACCATAA GGAAACGATA CAAAGAGCGA TATGGAAAATCCCTATTTCA TGATATCAGA AATTTTGCTT CAGGGCATTA TAAGAAAGCACTGCTTGCCA TCTGTGCTGG TGATGCTGAG GACTACTAAA ATGAAGAGGACTTGGAGTAC TGTGCACTCC TCTTTCTAGA CACTTCCAAA TAGAGATTTTCTCACAAATT TGTACTGTTC ATGGCACTAT TAACAAAACT ATACAATCATATTTTCTCTT CTATCTTTGA AATTATTCTA AGCCAAAGAA AACTATGAATGAAAGTATAT GATACTGAAT TTGCCTACTA TCCTGAATTT GCCTACTATCTAATCAGCAA TTAAATAAAT TGTGCATGAT GGAATAATAG AAAAATTGCATTGGAATAGA TTTTATTTAA ATGTGAACCA TCAACAACCT ACAACAA 7 Human ABLGGTTGGTGAC TTCCACAGGA AAAGTTCTGG AGGAGTAGCC AAAGACCATC mRNAAGCGTTTCCT TTATGTGTGA GAATTGAAAT GACTAGCATT ATTGACCCTTTTCAGCATCC CCTGTGAATA TTTCTGTTTA GGTTTTTCTT CTTGAAAAGAAATTGTTATT CAGCCCGTTT AAAACAAATC AAGAAACTTT TGGGTAACATTGCAATTACA TGAAATTGAT AACCGCGAAA ATAATTGGAA CTCCTGCTTGCAAGTGTCAA CCTAAAAAAA GTGCTTCCTT TTGTTATGGA AGATGTCTTTCTGTGATTGA CTTCAATTGC TGACTTGTGG AGATGCAGCG AATGTGAAATCCCACGTATA TGCCATTTCC CTCTACGCTC GCTGACCGTT CTGGAAGATCTTGAACCCTC TTCTGGAAAG GGGTACCTAT TATTACTTTA TGGGGCAGCAGCCTGGAAAA GTACTTGGGG ACCAAAGAAG GCCAAGCTTG CCTGCCCTGCATTTTATCAA AGGAGCAGGG AAGAAGGAAT CATCGAGGCA TGGGGGTCCACACTGCAATG TTTTTGTGGA ACATGAAGCC CTTCAGCGGC CAGTAGCATCTGACTTTGAG CCTCAGGGTC TGAGTGAAGC CGCTCGTTGG AACTCCAAGGAAAACCTTCT CGCTGGACCC AGTGAAAATG ACCCCAACCT TTTCGTTGCACTGTATGATT TTGTGGCCAG TGGAGATAAC ACTCTAAGCA TAACTAAAGGTGAAAAGCTC CGGGTCTTAG GCTATAATCA CAATGGGGAA TGGTGTGAAGCCCAAACCAA AAATGGCCAA GGCTGGGTCC CAAGCAACTA CATCACGCCAGTCAACAGTC TGGAGAAACA CTCCTGGTAC CATGGGCCTG TGTCCCGCAATGCCGCTGAG TATCTGCTGA GCAGCGGGAT CAATGGCAGC TTCTTGGTGCGTGAGAGTGA GAGCAGTCCT GGCCAGAGGT CCATCTCGCT GAGATACGAAGGGAGGGTGT ACCATTACAG GATCAACACT GCTTCTGATG GCAAGCTCTACGTCTCCTCC GAGAGCCGCT TCAACACCCT GGCCGAGTTG GTTCATCATCATTCAACGGT GGCCGACGGG CTCATCACCA CGCTCCATTA TCCAGCCCCAAAGCGCAACA AGCCCACTGT CTATGGTGTG TCCCCCAACT ACGACAAGTGGGAGATGGAA CGCACGGACA TCACCATGAA GCACAAGCTG GGCGGGGGCCAGTACGGGGA GGTGTACGAG GGCGTGTGGA AGAAATACAG CCTGACGGTGGCCGTGAAGA CCTTGAAGGA GGACACCATG GAGGTGGAAG AGTTCTTGAAAGAAGCTGCA GTCATGAAAG AGATCAAACA CCCTAACCTG GTGCAGCTCCTTGGGGTCTG CACCCGGGAG CCCCCGTTCT ATATCATCAC TGAGTTCATGACCTACGGGA ACCTCCTGGA CTACCTGAGG GAGTGCAACC GGCAGGAGGTGAACGCCGTG GTGCTGCTGT ACATGGCCAC TCAGATCTCG TCAGCCATGGAGTACCTGGA GAAGAAAAAC TTCATCCACA GAGATCTTGC TGCCCGAAACTGCCTGGTAG GGGAGAACCA CTTGGTGAAG GTAGCTGATT TTGGCCTGAGCAGGTTGATG ACAGGGGACA CCTACACAGC CCATGCTGGA GCCAAGTTCCCCATCAAATG GACTGCACCC GAGAGCCTGG CCTACAACAA GTTCTCCATCAAGTCCGACG TCTGGGCATT TGGAGTATTG CTTTGGGAAA TTGCTACCTATGGCATGTCC CCTTACCCGG GAATTGACCT GTCCCAGGTG TATGAGCTGCTAGAGAAGGA CTACCGCATG GAGCGCCCAG AAGGCTGCCC AGAGAAGGTCTATGAACTCA TGCGAGCATG TTGGCAGTGG AATCCCTCTG ACCGGCCCTCCTTTGCTGAA ATCCACCAAG CCTTTGAAAC AATGTTCCAG GAATCCAGTATCTCAGACGA AGTGGAAAAG GAGCTGGGGA AACAAGGCGT CCGTGGGGCTGTGAGTACCT TGCTGCAGGC CCCAGAGCTG CCCACCAAGA CGAGGACCTCCAGGAGAGCT GCAGAGCACA GAGACACCAC TGACGTGCCT GAGATGCCTCACTCCAAGGG CCAGGGAGAG AGCGATCCTC TGGACCATGA GCCTGCCGTGTCTCCATTGC TCCCTCGAAA AGAGCGAGGT CCCCCGGAGG GCGGCCTGAATGAAGATGAG CGCCTTCTCC CCAAAGACAA AAAGACCAAC TTGTTCAGCGCCTTGATCAA GAAGAAGAAG AAGACAGCCC CAACCCCTCC CAAACGCAGCAGCTCCTTCC GGGAGATGGA CGGCCAGCCG GAGCGCAGAG GGGCCGGCGAGGAAGAGGGC CGAGACATCA GCAACGGGGC ACTGGCTTTC ACCCCCTTGGACACAGCTGA CCCAGCCAAG TCCCCAAAGC CCAGCAATGG GGCTGGGGTCCCCAATGGAG CCCTCCGGGA GTCCGGGGGC TCAGGCTTCC GGTCTCCCCACCTGTGGAAG AAGTCCAGCA CGCTGACCAG CAGCCGCCTA GCCACCGGCGAGGAGGAGGG CGGTGGCAGC TCCAGCAAGC GCTTCCTGCG CTCTTGCTCCGCCTCCTGCG TTCCCCATGG GGCCAAGGAC ACGGAGTGGA GGTCAGTCACGCTGCCTCGG GACTTGCAGT CCACGGGAAG ACAGTTTGAC TCGTCCACATTTGGAGGGCA CAAAAGTGAG AAGCCGGCTC TGCCTCGGAA GAGGGCAGGGGAGAACAGGT CTGACCAGGT GACCCGAGGC ACAGTAACGC CTCCCCCCAGGCTGGTGAAA AAGAATGAGG AAGCTGCTGA TGAGGTCTTC AAAGACATCATGGAGTCCAG CCCGGGCTCC AGCCCGCCCA ACCTGACTCC AAAACCCCTCCGGCGGCAGG TCACCGTGGC CCCTGCCTCG GGCCTCCCCC ACAAGGAAGAAGCTGGAAAG GGCAGTGCCT TAGGGACCCC TGCTGCAGCT GAGCCAGTGACCCCCACCAG CAAAGCAGGC TCAGGTGCAC CAGGGGGCAC CAGCAAGGGCCCCGCCGAGG AGTCCAGAGT GAGGAGGCAC AAGCACTCCT CTGAGTCGCCAGGGAGGGAC AAGGGGAAAT TGTCCAGGCT CAAACCTGCC CCGCCGCCCCCACCAGCAGC CTCTGCAGGG AAGGCTGGAG GAAAGCCCTC GCAGAGCCCGAGCCAGGAGG CGGCCGGGGA GGCAGTCCTG GGCGCAAAGA CAAAAGCCACGAGTCTGGTT GATGCTGTGA ACAGTGACGC TGCCAAGCCC AGCCAGCCGGGAGAGGGCCT CAAAAAGCCC GTGCTCCCGG CCACTCCAAA GCCACAGTCCGCCAAGCCGT CGGGGACCCC CATCAGCCCA GCCCCCGTTC CCTCCACGTTGCCATCAGCA TCCTCGGCCC TGGCAGGGGA CCAGCCGTCT TCCACCGCCTTCATCCCTCT CATATCAACC CGAGTGTCTC TTCGGAAAAC CCGCCAGCCTCCAGAGCGGA TCGCCAGCGG CGCCATCACC AAGGGCGTGG TCCTGGACAGCACCGAGGCG CTGTGCCTCG CCATCTCTAG GAACTCCGAG CAGATGGCCAGCCACAGCGC AGTGCTGGAG GCCGGCAAAA ACCTCTACAC GTTCTGCGTGAGCTATGTGG ATTCCATCCA GCAAATGAGG AACAAGTTTG CCTTCCGAGAGGCCATCAAC AAACTGGAGA ATAATCTCCG GGAGCTTCAG ATCTGCCCGGCGACAGCAGG CAGTGGTCCA GCGGCCACTC AGGACTTCAG CAAGCTCCTCAGTTCGGTGA AGGAAATCAG TGACATAGTG CAGAGGTAGC AGCAGTCAGGGGTCAGGTGT CAGGCCCGTC GGAGCTGCCT GCAGCACATG CGGGCTCGCCCATACCCGTG ACAGTGGCTG ACAAGGGACT AGTGAGTCAG CACCTTGGCCCAGGAGCTCT GCGCCAGGCA GAGCTGAGGG CCCTGTGGAG TCCAGCTCTACTACCTACGT TTGCACCGCC TGCCCTCCCG CACCTTCCTC CTCCCCGCTCCGTCTCTGTC CTCGAATTTT ATCTGTGGAG TTCCTGCTCC GTGGACTGCAGTCGGCATGC CAGGACCCGC CAGCCCCGCT CCCACCTAGT GCCCCAGACTGAGCTCTCCA GGCCAGGTGG GAACGGCTGA TGTGGACTGT CTTTTTCATTGAGCTCTCCA GGCCAGGTGG GAACGGCTGA TGTGGACTGT CTTTTTCATTTTTTTCTCTC TGGAGCCCCT CCTCCCCCGG CTGGGCCTCC TTCTTCCACTTCTCCAAGAA TGGAAGCCTG AACTGAGGCC TTGTGTGTCA GGCCCTCTGCCTGCACTCCC TGGCCTTGCC CGTCGTGTGC TGAAGACATG TTTCAAGAACCGCATTTCGG GAAGGGCATG CACGGGCATG CACACGGCTG GTCACTCTGCCCTCTGCTGC TGCCCGGGGT GGGGTGCACT CGCCATTTCC TCACGTGCAGGACAGCTCTT GATTTGGGTG GAAAACAGGG TGCTAAAGCC AACCAGCCTTTGGGTCCTGG GCAGGTGGGA GCTGAAAAGG ATCGAGGCAT GGGGCATGTCCTTTCCATCT GTCCACATCC CCAGAGCCCA GCTCTTGCTC TCTTGTGACGTGCACTGTGA ATCCTGGCAA GAAAGCTTGA GTCTCAAGGG TGGCAGGTCACTGTCACTGC CGACATCCCT CCCCCAGCAG AATGGAGGCA GGGGACAAGGGAGGCAGTGG CTAGTGGGGT GAACAGCTGG TGCCAAATAG CCCCAGACTGGGCCCAGGCA GGTCTGCAAG GGCCCAGAGT GAACCGTCCT TTCACACATCTGGGTGCCCT GAAAGGGCCC TTCCCCTCCC CCACTCCTCT AAGACAAAGTAGATTCTTAC AAGGCCCTTT CCTTTGGAAC AAGACAGCCT TCACTTTTCTGAGTTCTTGA AGCATTTCAA AGCCCTGCCT CTGTGTAGCC GCCCTGAGAGAGAATAGAGC TGCCACTGGG CACCTGCGCA CAGGTGGGAG GAAAGGGCCTGGCCAGTCCT GGTCCTGGCT GCACTCTTGA ACTGGGCGAA TGTCTTATTTAATTACCGTG AGTGACATAG CCTCATGTTC TGTGGGGGTC ATCAGGGAGGGTTAGGAAAA CCACAAACGG AGCCCCTGAA AGCCTCACGT ATTTCACAGAGCACGCCTGC CATCTTCTCC CCGAGGCTGC CCCAGGCCGG AGCCCAGATACGGGGGCTGT GACTCTGGGC AGGGACCCGG GGTCTCCTGG ACCTTGACAGAGCAGCTAAC TCCGAGAGCA GTGGGCAGGT GGCCGCCCCT GAGGCTTCACGCCGGGAGAA GCCACCTTCC CACCCCTTCA TACCGCCTCG TGCCAGCAGCCTCGCACAGG CCCTAGCTTT ACGCTCATCA CCTAAACTTG TACTTTATTTTTCTGATAGA AATGGTTTCC TCTGGATCGT TTTATGCGGT TCTTACAGCACATCACCTCT TTGCCCCCGA CGGCTGTGAC GCAGCCGGAG GGAGGCACTAGTCACCGACA GCGGCCTTGA AGACAGAGCA AAGCGCCCAC CCAGGTCCCCCGACTGCCTG TCTCCATGAG GTACTGGTCC CTTCCTTTTG TTAACGTGATGTGCCACTAT ATTTTACACG TATCTCTTGG TATGCATCTT TTATAGACGCTCTTTTCTAA GTGGCGTGTG CATAGCGTCC TGCCCTGCCC CCTCGGGGGCCTGTGGTGGC TCCCCCTCTG CTTCTCGGGG TCCAGTGCAT TTTGTTTCTGTATATGATTC TCTGTGGTTT TTTTTGAATC CAAATCTGTC CTCTGTAGTATTTTTTAAAT AAATCAGTGT TTACATTAGA A 8 Armored RNA ®GAUGCUCACU UCAUCUAUGG UUACCCUGGG ACUUUUACAC CAACAGAACU sequenceAGCAUCAUCC UCUGCAUGGU CAGGUCAUGG AUCGGCAUCC UGACAGUUUCGGGAAUUAGG CAUCUGCAGU CUUACUGCUC AUCGGCUGAU GAUGCUGCUGUAAUCCCCAU CCAAGCAAGC UUGUGAUCCU CCGCCAUUAU CCCAAAUGGUAUAACAUUUA GGACUUAAAG CUAUGCAAUU AUCACCUUGU UUUUCAACAGCAAGACCUAA UAUUUUCUUU UCAUCAUUAA UGCCUUUUGA UGGAUCAGGCAACCAUUUAU AAAUAUGUUC ACCAGCCGAA GUCAGUAGUG AUUGGGUGGUUCCUGGCUUG GGAUCAUGCC GCUGCAGAGG CUAUUCUCCU CUUGGCAGAUUGUCUGUAGC CGAGAAGGCG GAGUCUGGCA AUGAUCAUGC AUACAGUGUACGACAGCCUU AGGGACUGGA GCUCAAGCAG UGUUUCCUCA ACCAGUCACA

1. A method for detecting a set of bladder cancer markers in a sample from the subject comprising: (a) obtaining a urine sample or bladder washing sample from the subject; (b) contacting RNA from the sample with a set of primers, wherein the set of primers consists of i. a first and second primer for detecting corticotrophin releasing hormone (CRH), ii. a first and second primer for detecting insulin-like growth factor 2 (IGF2), iii. a first and second primer for detecting keratin 20 (KRT20), and iv. a first and second primer for detecting annexin 10 (ANXA10); and (c) detecting hybridization between the RNA and the set of primers.
 2. The method of claim 1, wherein the method further comprises detecting an endogenous control and/or exogenous control.
 3. The method of claim 2, wherein the endogenous control is selected from ABL, GUSB, GAPDH, TUBB, and UPK1.
 4. (canceled)
 5. The method of claim 2, wherein the exogenous control is an RNA.
 6. The method of claim 1, wherein the detecting comprises RT-PCR.
 7. (canceled)
 8. The method of claim 2, wherein the method comprises comparing a Ct value or a ΔCt value to a threshold Ct value or ΔCt value, wherein ΔCt is the Ct value for the control minus the Ct value for the marker.
 9. (canceled)
 10. The method of claim 1, wherein the detecting comprises a RT-PCR reaction that takes less than 2 hours from an initial denaturation step through a final extension step. 11.-14. (canceled)
 15. The method of claim 1, wherein the first and second primer for detecting CRH comprise: a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 19 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 20, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 35 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 36, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 16. The method of claim 1, wherein the first and second primer for detecting IGF2 comprise: a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 16 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 17, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or b) a first primer comprising at least 12 contiguous nucleotides of at least 12 contiguous nucleotides of SEQ ID NO: 32 and a second primer comprising SEQ ID NO: 33, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 17. The method of claim 1, wherein the first and second primer for detecting KRT20 comprise: a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 13 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 14, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 29 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 30, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 18. The method of claim 1, wherein the first and second primer for detecting ANXA10 comprise: a) comprises a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 26 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 27, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 38 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 39, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or c) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 48 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 39, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long. 19.-22. (canceled)
 23. The method of claim 1, wherein the method further comprises forming a set of bladder cancer marker amplicons, wherein the set of bladder cancer marker amplicons consists of a CRH amplicon, an IGF2 amplicon, a KRT20 amplicon, and an ANXA10 amplicon, and contacting the bladder cancer marker amplicons with a set of bladder cancer marker probes, wherein the set of bladder cancer marker probes consists of a first probe for detecting the CRH amplicon, a second probe for detecting the IGF2 amplicon, a third probe for detecting the KRT20 amplicon, and a fourth probe for detecting the ANXA10 amplicon.
 24. The method of claim 23, wherein the first probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 21 or SEQ ID NO: 37, wherein the first probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 25. The method of claim 23, wherein the second probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 34 or at least 12 SEQ ID NO: 18, wherein the second probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 26. The method of claim 23, wherein the third probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 15 or SEQ ID NO: 31, wherein the third probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 27. The method of claim 23, wherein the fourth probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 28 or SEQ ID NO: 40, wherein the fourth probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.
 28. The method of claim 23, wherein each bladder cancer marker probe comprises a dye, and wherein each dye is detectably different from the other three dyes.
 29. (canceled)
 30. The method of claim 23, wherein the method further comprises forming an endogenous control amplicon, and contacting the endogenous control amplicon with an endogenous control probe, and/or forming an exogenous control amplicon, and contacting the exogenous control amplicon with an exogenous control probe, wherein each probe comprises a dye, and wherein each dye is detectably different from the other dyes. 31.-41. (canceled)
 42. A composition comprising a set of bladder cancer marker primer pairs, wherein the set of bladder cancer marker primer pairs consists of a first primer pair for detecting CRH, a second primer pair for detecting IGF2, a third primer pair for detecting KRT20, and a fourth primer pair for detecting ANXA10. 43.-61. (canceled)
 62. A kit comprising the composition of claim
 42. 